MODIFIED GUIDE RNAs

Abstract

The present disclosure relates to CRISPR-related genome editing systems having a gRNA molecule with one or more modifications for editing and / or modulating the expression of a target nucleic acid sequence of interest. The present disclosure is also directed to methods and applications thereof in connection with the treatment and / or management of disease using the described genome editing systems and gRNA molecules.

Description

[0001] MODIFIED GUIDE RNAs

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priori ty to U.S. Provisional Application No. 63 / 549,247, filed February 2, 2024, U.S. Provisional Application No. 63 / 557,142, filed on February 23, 2024, U.S. Provisional Application No. 63 / 631,436, filed April 8, 2024, U.S. Provisional Application No. 63 / 643,353, filed May 6, 2024, and U.S. Provisional Application No. 63 / 718,386, filed November 8, 2024 the contents of each of which are incorporated by reference in their entirety , and to each of which priority is claimed.

[0004] SEQUENCE LISTING

[0005] The present specification makes reference to a Sequence Listing (submitted electronically as a xml file named "0841770309_ST26.xml" on February 2, 2025). The 0841770309_ST26.xml file was generated on January 31, 2025, and is 775,596 bytes in size. The entire contents of the Sequence Listing are hereby incorporated by reference.

[0006] FIELD

[0007] The present disclosure is directed to modified gRNAs and CRISPR-related genome editing systems and components for targeting, editing and / or modulating the expression of a target nucleic acid sequence of interest. The present disclosure is also directed to methods and applications thereof in connection with the treatment and / or management of disease.

[0008] BACKGROUND

[0009] CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) evolved in bacteria and archaea as an adaptive immune system to defend against viral attack. Upon exposure to a virus, short segments of viral DNA are integrated into the CRISPR locus. RNA is transcribed from a portion of the CRISPR locus that includes the viral sequence. That RNA, which contains sequence complementary to the viral genome, mediates targeting of a Cas protein to a target sequence in the viral genome. The Cas protein, in turn, cleaves and thereby silences the viral target. Naturally occurring CRISPR systems are organized evolutionarily into two classes and five types. Casl2a (also known as Cpfl) represents a Class 2. Type V CRISPR / Cas system that has been adapted for genome editing in eukaryotic cells. The introduction of site- specific double strand breaks (DSBs) into the targeted sequence allows for knocking out a gene through the formation of an indel through endogenous DNA repair mechanisms, for example non-homologous end-joining (NHEJ). The introduction of site-specific DSBs into the targeted sequence can also facilitate gene conversion or gene correction through the incorporation of an exogenous or endogenous homologous sequence with a repair template, for example by homology -directed repair (HDR).

[0010] SUMMARY

[0011] The presently disclosed subject matter is directed, in certain embodiments, to a genome editing system comprising (a) a gRNA molecule comprising a targeting domain that targets a target sequence of a gene of interest, wherein the gRNA molecule contains one or more modifications, and (b) an RNA-guided nuclease, or a nucleic acid (e.g., an RNA) encoding the RNA-guided nuclease thereof.

[0012] In certain embodiments, the gRNA molecule of the genome editing system further comprises a nucleotide extension. In certain embodiments, the nucleotide extension is a 5' extension, a 3’ extension, or any combination thereof. In certain embodiments, the nucleotide extension comprises one or more RNA bases, one or more DNA bases, or a combination thereof. In certain embodiments, the gRNA molecule of the genome editing system comprises one or more modifications. In certain embodiments, the one or more modifications is selected from the group consisting of a 5’ inverted thymidine (idT) modification, a 3’ idT modification, a 2’ fluoro modification, a 2’ O-methyl modification, a phosphorothioate linkage, a 3’ pseudoknot, a locked nucleic acid (LNA), and any combination thereof. In certain embodiments, the one or more modifications comprises the 5’ inverted thymidine (idT) modification and the 3’ idT modification. In certain embodiments, the one or more modifications comprises the 2’ fluoro modification. In certain embodiments, the one or more modifications comprises one or more 2’ fluoro modifications, and each of the 2’ fluoro modifications modifies a nucleotide internal to the gRNA molecule. In certain embodiments, the one or more modifications of the gRNA of the genome editing system enhance binding affinity of the gRNA molecule to the RNA-guided nuclease, e g., a Cas 12a nuclease, of the genome editing system.

[0013] In certain embodiments, the RNA-guided nuclease of the genome editing system comprises AsCasl2a. In certain embodiments, the gRNA molecule comprises a hairpin region capable of binding to the AsCasl2a. In certain embodiments, the one or more modifications of the gRNA are in the hairpin region, the targeting domain, or both. In certain embodiments, the one or more modifications of the gRNA comprises a 5’ inverted thymidine (idT) modification and / or a 3’ idT modification. In certain embodiments, the gRNA molecule comprises a DNA extension 5’ to the hairpin region. In certain embodiments, the hairpin region is upstream of the targeting domain.

[0014] In certain embodiments, at least one of nucleotides 1. 5, 6, 7, 8, 9, 10, 12, 13, 14. 16, 17, 18 or 19 of the hairpin region is modified with a 2’ fluoro modification. In certain embodiments, each of nucleotides 1 , 5, 6, 7, 8, 9, 10, 12, 13, 14, 16, 17, 18 and 19 of the hairpin region is modified with a 2’ fluoro modification. In certain embodiments, a pattern of 2’ fluoro modifications on the hairpin region of the gRNA consists of 2’ fluoro modifications at nucleotides 1, 5. 6, 7, 8. 9, 10, 12. 13. 14. 16. 17, 18 and 19 of the hairpin region. In certain embodiments, nucleotides 7 and 8 of the hairpin region are not modified with a 2’ fluoro modification. In certain embodiments, a pattern of 2’ fluoro modifications on the hairpin region of the gRNA consists of 2’ fluoro modifications at nucleotides 1, 5, 6, 9, 10, 12, 13, 14, 16, 17, 18 and 19 of the hairpin region.

[0015] In certain embodiments, the one or more modifications of the gRNA enhance binding affinity of the gRNA molecule to the Cas 12a nuclease.

[0016] In certain embodiments, the hairpin region comprises SEQ ID NO: 252. In certain embodiments, the hairpin region comprises SEQ ID NO: 421. In certain embodiments, the hairpin region comprises SEQ ID NO: 427.

[0017] In certain embodiments, the subject is a human subject.

[0018] In certain embodiments, the gene of interest is expressed in a liver cell, e.g., a hepatocyte) of a subject.

[0019] In certain embodiments, the liver-expressed gene is LPA. ANGPT1.3. PCSK9, LDLR, APOC2, APOC3. APOB, MTP, ANGPTL4, ANGPTL8, APOA5, ApoB, APOE, IDOL, NPC1L1, ASGR1, TM6SF2, GALNT2, LPL, MLXIPL, SORT1, TRIBI, MARC1, ABCG5, ABCG8, PNPLA3, TM6SF2, HFE, GCKR, HMOX-1. UGT1A1, STAP1, LDLRAP1. LMF-1, GP1HBP1, CYP27A1, LIPA orATP7B.

[0020] In certain embodiments, the liver-expressed gene is involved in regulating lipid and / or cholesterol metabolism. In certain embodiments, decreasing the expression of the liver-expressed gene decreases the levels of circulating LDL. In certain embodiments, decreasing the expression of the liver-expressed gene decreases the levels of circulating Lp(a). In certain embodiments, the liver-expressed gene is LPA, ANGPTL3, PCSK9, LDLR, APOC2, APOC3, APOB, MTP, ANGPTL4, ANGPTL8, APOA5, ApoB, APOE, IDOL, NPC1L1, ASGR1, TM6SF2, GALNT2, LPL, MLXIPL, SORT1, TRIBI, MARC1, ABCG5, ABCG8, PNPLA3, TM6SF2, HFE, HMOX-1, UGT1A1, STAP1, LDLRAP1, LMF-1, GP1HBP1, CYP27A1, LIPA wATP7B.

[0021] In certain embodiments, the gene of interest is expressed in a kidney cell (e.g., a renal epithelial cell) of a subject.

[0022] In certain embodiments, the gene of interest is expressed in a stem cell (e.g., a CD34+hematopoietic stem and progenitor cell) of a subject.

[0023] In certain embodiments, the gene of interest is expressed in an ocular cell (e.g., a trabecular mesh work cell) of a subject. In certain embodiments, the gene of interest is MYOC.

[0024] The presently disclosed subject matter is directed, in certain embodiments, to a genome editing system comprising (a) a gRNA molecule comprising a targeting domain that targets a sequence of a Proprotein convertase subtilisn / kexin ty pe 9 (PCSK9) gene, and (b) an RNA-guided nuclease, or a nucleic acid (e.g., an RNA) encoding the RNA-guided nuclease.

[0025] In certain embodiments, the target sequence of the PCSK9 gene comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 321-335. In certain embodiments, the target sequence of the PCSK9 gene comprises the nucleotide sequence set forth in SEQ ID NO: 344, or SEQ ID NO: 335. In certain embodiments, the targeting domain comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 347-361. In certain embodiments, the targeting domain comprises the nucleotide sequence set forth in SEQ ID NO: 360, or SEQ ID NO: 361.

[0026] In certain embodiments, the RNA-guided nuclease is a Casl2a protein. In certain embodiments, the RNA-guided nuclease is a modified Casl2a protein. In certain embodiments, the modified Casl2a protein is an activity enhanced Casl2a protein. In certain embodiments, the RNA-guided nuclease comprises the amino acid sequence set forth in of any one of SEQ ID NOs: 72-79. 423, 424 or 430-432. In certain embodiments, the nucleic acid encoding the RNA-guided nuclease is an RNA. In certain embodiments, the RNA encoding the RNA-guided nuclease comprises a nucleotide sequence selected from the group consisting of SEQ ID Nos: 93-97. In certain embodiments, the RNA encoding the RNA-guided nuclease comprises the nucleotide sequence of SEQ ID NO: 97 or any one of SEQ ID NO: 433-437. In certain embodiments, the RNA-guided nuclease is an AsCasl2a, for example, an AsCasl2a comprising the amino acid sequence set forth in SEQ ID NO: 75.

[0027] The presently disclosed subject matter is also directed to a ribonucleoprotein (RNP) complex comprising the genome editing system disclosed herein.

[0028] The presently disclosed subject matter is also directed to a delivery system for delivering the genome editing system disclosed herein.

[0029] In certain embodiments, the delivery' system comprises a DNA sequence encoding the gRNA molecule.

[0030] In certain embodiments, the delivery system comprises a DNA sequence encoding the RNA-guided nuclease. In certain embodiments, the delivery system comprises an RNA sequence encoding the gRNA molecule.

[0031] In certain embodiments, the delivery system comprises an RNA sequence encoding the RNA-guided nuclease.

[0032] In certain embodiments, the delivery system comprises a lipid nanoparticle (LNP).

[0033] In certain embodiments, the LNP comprises an ionizable lipid, a PEG lipid, a helper lipid, a sterol, or any combination thereof.

[0034] In certain embodiments, the ionizable lipid is selected from the group consisting of ((4-Hydroxybutyl)azanediyl)bis(hexane-6,l-diyl) bis(2-hexyldecanoate) (ALC- 0315), and 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1- octylnonyl ester (SM-102), (2Z)-2-Nonen-l-yl 4-[[[[2- (dimethylamino)ethyl]thio]carbonyl] [4-[(l-heptyloctyl)oxy]-4- oxobutyl] amino] butanoate (ATX-081), ATX-095 and ATX-0126.

[0035] In certain embodiments, the PEG lipid is selected from the group consisting of dimyristoyl-sn-glycero-3-methoxypolyethylene glycol (DMG-PEG), distearoyl-sn- glycerol-3-methoxypolyethylene glycol (DSG-PEG), and distearoyl-sn-glycero-3- phosphoethanolamine-N-methoxypolyethylene glycol (DSPE-PEG).

[0036] In certain embodiments, the helper lipid is selected from the group consisting of distearoylphosphatidylcholine (DSPC) and l,2-dioleoyl-sn-glycero-3- phosphoethanolamine. (DOPE). In certain embodiments, the sterol is selected from the group consisting of cholesterol and sitosterol.

[0037] In certain embodiments, the LNP comprises ((4- Hydroxybutyl)azanediyl)bis(hexane-6,l-diyl) bis(2 -hexyldecanoate) (ALC-0315), DMG-PEG, DSPC, and cholesterol.

[0038] The presently disclosed subject matter is also directed to a method of editing a gene of interest in a target cell comprising contacting the target cell with the genome editing system, the RNP complex, and / or the delivery system disclosed herein.

[0039] In certain embodiments, the target cell is in vivo. In certain embodiments, the target cell is a cell involved in metabolism. In certain embodiments, the target cell is a hepatocyte.

[0040] The presently disclosed subject matter is also directed to a method of treating a disease or disorder comprising administering to a subject in need thereof, the genome editing system, the RNP complex, and / or the delivery system disclosed herein.

[0041] In certain embodiments, the disease or disorder is a hyperlipidemia or hypercholesterolemia. In certain embodiments, the disease or disorder is homozygous familial hypercholesterolemia (HoFH) or heterozygous familial hypercholesterolemia (HeFH). In certain embodiments, the subject is suffering from an atherosclerotic cardiovascular disease (ASCVD). In certain embodiments, the subject is identified to be at a high risk for a major adverse cardiovascular event (MACE) or has suffered from a MACE. In certain embodiments, the subject has an Apolipoprotein(a) (Apo(a)) or Lp(a) level (e.g., a serum or plasma level) >150 mg / dL.

[0042] In certain embodiments, administering the genome editing system, the RNP complex, or the delivery system to the subject reduces the level of a gene product of the gene of interest in the subject, or in a cell, tissue, or fluid from the subject, by at least about 50% to at least about 95% relative to the level prior to administration (or relative to the level in a control subject, cell, tissue, or fluid). In certain embodiments, administering the genome editing system, the RNP complex, or the delivery system to the subject reduces the level of a gene product of the gene of interest in the subject, or in a cell, tissue, or fluid from the subject, by at least about 90% relative to the level prior to administration (or relative to the level in a control subject, cell, tissue, or fluid). In certain embodiments, administering the genome editing system, the RNP complex, or the delivery system reduces the level of a gene product of the gene of interest in a cell from the subject by at least about 50% to at least about 95% relative to the expression level prior to administration (or relative to the level in a control). In certain embodiments, administering the genome editing system, the RNP complex, or the delivery system to the subject reduces the level of a gene product of the gene of interest in the subject, or in a cell, tissue, or fluid of the subject by at least about 90% relative to the level prior to administration (or relative to the level in a control).

[0043] The presently disclosed subject matter is also directed to a method of treating a disease or disorder comprising administering to a subject in need thereof, the genome editing system, the RNP complex, and / or the delivery system disclosed herein, further comprising, administering to the subject a standard of care (SOC) for hyperlipidemia or hypercholesterolemia. In certain embodiments, the SOC is Apo(a) apheresis and / or at least one pharmacological agent. In certain embodiments, the pharmacological agent is selected from the group consisting of a statin, an angiopoietin like 3 (ANGPTL3) inhibitor, a proprotein convertase subtilisin / kexin type 9 (PCSK9) inhibitor, LY3473329, and any combination thereof.

[0044] The presently disclosed subject matter is also directed to a method of treating a disease in a subject in need thereof, the method comprising administering to the subject a formulation comprising a lipid nanoparticle (LNP), an mRNA encoding a Cas l2a nuclease, and a gRNA molecule comprising a targeting domain that targets a sequence of a gene of interest of the subject, wherein the gRNA molecule contains one or more modifications and wherein the mRNA and the gRNA are encapsulated within the LNP.

[0045] In certain embodiments, the gRNA molecule further comprises a nucleotide extension. In certain embodiments, the nucleotide extension is a 5’ extension, a 3’ extension, or a combination thereof. In certain embodiments, the nucleotide extension comprises one or more RNA bases, one or more DNA bases, or any combination thereof. In certain embodiments, the gRNA molecule contains one or more modifications. In certain embodiments, the one or more modifications is selected from the group consisting of a 5’ inverted thymidine (idT) modification, a 3’ idT modification, a 2’ fluoro modification, a 2’ O-methyl modification, a phosphorothioate linkage, a 3‘ pseudoknot, a locked nucleic acid (LNA) and any combination thereof. In certain embodiments, the one or more modifications comprises the 5’ inverted thymidine (idT) modification and the 3’ idT modification. In certain embodiments, the one or more modifications comprises the 2’ fluoro modification. In certain embodiments, the one or more modifications comprises one or more 2" fluoro modifications, and each of the 2‘ fluoro modifications modifies a nucleotide internal to the gRNA molecule.

[0046] In certain embodiments, the one or more modifications of the gRNA are in the hairpin region, the targeting domain, or both. In certain embodiments, the hairpin region is upstream of the targeting domain. In certain embodiments, the hairpin region comprises the one or more modifications. In certain embodiments, the hairpin region comprises SEQ ID NO: 252. In certain embodiments, the hairpin region comprises SEQ ID NO: 421 . In certain embodiments, the hairpin region comprises SEQ ID NO: 427.

[0047] In certain embodiments, at least one of nucleotides 1, 5, 6, 7, 8, 9, 10, 12, 13, 14, 16, 17, 18 or 19 of the hairpin region is modified with a 2’ fluoro modification. In certain embodiments, each of nucleotides 1, 5, 6, 7, 8. 9, 10, 12, 13, 14, 16. 17. 18 and 19 of the hairpin region is modified with a 2’ fluoro modification. In certain embodiments, a pattern of 2’ fluoro modifications on the hairpin region of the gRNA consists of 2’ fluoro modifications at nucleotides 1, 5, 6, 7, 8, 9, 10, 12, 13, 14, 16, 17, 18 and 19 of the hairpin region. In certain embodiments, nucleotides 7 and 8 of the hairpin region are not modified with a 2’ fluoro modification. In certain embodiments, a pattern of 2’ fluoro modifications on the hairpin region of the gRNA consists of 2’ fluoro modifications at nucleotides 1, 5, 6, 9, 10, 12, 13, 14, 16, 17, 18 and 19 of the hairpin region.

[0048] In certain embodiments, the one or more modifications of the gRNA enhance binding affinity of the gRNA molecule to the Cas 12a nuclease.

[0049] In certain embodiments, the gRNA molecule comprises a DNA extension 5’ to the gene of interest is expressed in a liver cell of a subject. In certain embodiments, the subject is a human subject.

[0050] In certain embodiments, the disease or disorder is a hyperlipidemia or hypercholesterolemia. In certain embodiments, the disease or disorder is homozygous familial hypercholesterolemia (HoFH) or heterozygous familial hypercholesterolemia (HeFH).

[0051] In certain embodiments of any of the foregoing methods, the gene of interest is expressed in a liver cell (e.g., a hepatocyte) of a subject.

[0052] In certain embodiments, the liver-expressed gene is LPA, ANGPTL3, PCSK9, LDLR, APOC2, APOC3, APOB, MTP, ANGPTL4, ANGPTL8, APOA5, ApoB, APOE, IDOL, NPC1L1, ASGR1, TM6SF2, GALNT2, LPL, MLXIPL. SORTI, TRIBI, MARC1, ABCG5, ABCG8, PNPM3, TM6SF2, HFE, GCKR, HMOX-1, UGT1A1, STAP1. LDLRAP1. LMF-1, GP1HBP1, CYP27A1, LIPA or ATP7B.

[0053] In certain embodiments, the liver-expressed gene is involved in regulating lipid and / or cholesterol metabolism. In certain embodiments, decreasing the expression of the liver-expressed gene decreases the levels of circulating LDL. In certain embodiments, decreasing the expression of the liver-expressed gene decreases the levels of circulating Lp(a). In certain embodiments, the liver-expressed gene is LPA, ANGPTL3, PCSK9, LDLR, APOC2, APOC3, APOB, MTP, ANGPTL4, ANGPTL8, APOA5, ApoB, APOE, IDOL, NPC1L1, ASGR1, TM6SF2, GALNT2, LPL, MLXIPL, SORT1, TRIBI, MARC1, ABCG5, ABCG8, PNPLA3, TM6SF2, HFE, HMOX-1, UGT1A1, STAP1, LDLRAP1, LMF-1, GP1HBP1, CYP27A1, LIPA orATP7B.

[0054] In certain embodiments, the gene of interest is expressed in a kidney cell (e.g.. a renal epithelial cell) of a subject.

[0055] In certain embodiments, the gene of interest is expressed in a stem cell (e.g., a CD34+hematopoietic stem and progenitor cell) of a subject.

[0056] In certain embodiments, the gene of interest is expressed in an ocular cell (e.g., a trabecular mesh work cell) of a subject. In certain embodiments, the gene of interest is MYOC.

[0057] The presently disclosed subject matter is also directed to a method of treating a disase or disorder (e.g., an atherosclerotic cardiovascular disease) in a subject in need thereof, the method comprising administering to the subject a formulation comprising a lipid nanoparticle (LNP), an mRNA encoding a Casl2a nuclease, and a gRNA molecule comprising a targeting domain that targets a sequence of a Proprotein convertase subtilisin / kexin type 9 (PCSK9) gene of the subject, wherein the mRNA and the gRNA are encapsulated within the LNP.

[0058] In certain embodiments, the target sequence of the PCSK9 gene comprises a nucleotide sequence selected from the group consisting of SEQ ID Nos: 321-335.

[0059] In certain embodiments, the targeting domain comprises a nucleotide sequence selected from the group consisting of SEQ ID Nos: 347-361. In certain embodiments, the gRNA molecule comprises the sequence set forth in SEQ ID NO: 360.

[0060] In certain embodiments, the gRNA molecule comprises the sequence and modifications set forth in SEQ ID NO: 391.

[0061] The presently disclosed subject matter is also directed to a gRNA molecule comprising a targeting domain that targets a sequence of a gene interest, wherein the gRNA molecule comprises one or more modifications selected from the group consisting of a 5’ inverted thymidine (idT) modification, a 3’ idT modification, a 2’ fluoro modification, a 2’ O-methyl modification, a phosphorothioate linkage and a combination thereof.

[0062] In certain embodiments, the one or more modifications are on nucleotides positioned outside of the targeting domain. In certain embodiments, the gRNA comprises a 5’ DNA extension. In certain embodiments, the 5’ DNA extension comprises or consists of the sequence set forth in SEQ ID NO: 7.

[0063] In certain embodiments, the gRNA molecule comprises the following sequence: / 5InvdT / ATGTGTTTTTGTCAAAAGACCTTTT / i2FU / rArArU / i2FU / / i2FU / / i2FC / / i2FU / / i2FA / / i2FC7rU / i2FC / / i2FU / / i2FU / rG / i2FU / / i2FA / / i2FG / / i2FA / rUx / 3InvdT / (SEQ ID NO: 113); wherein x comprises or consists of the targeting domain. In certain embodiments, the gRNA molecule comprises the following sequence: / 5InvdT / ATGTGTTTTTGTCAAAAGACCTTTT / i2FU / rArArU / i2FU / / i2FU / / i2FC / / i 2FU / / i2FA / / i2FC / rU / i2FC / / i2FU / / i2FU / rG / i2FU / / i2FA / / i2FG / / i2FA / rUx (SEQ ID NO: 438); wherein x comprises or consists of the targeting domain. In certain embodiments, the gRNA molecule comprises the following sequence: / i2FU / rArArU / i2FU / / i2FU / / i2FC / / i2FU / / i2FA / / i2FC / rU / i2FC / / i2FU / / i2FU / rG / i2FU / / i 2FA / / i2FG / / i2FA / rUx / 3InvdT / (SEQ ID NO: 439); wherein x comprises or consists of the targeting domain. In certain embodiments, the gRNA molecule comprises the following sequence:

[0064] / i2FU / rArArU / i2FU / / i2FU / / i2FC / / i2FU / / i2FA / / i2FC / rU / i2FC / / i2FU / / i2FU / rG / i2FU / / i 2FA / / i2FG / / i2FA / rUx (SEQ ID NO: 440); wherein x comprises or consists of the targeting domain,.

[0065] In certain embodiments, the gRNA molecule comprises the following sequence: mA*TGTGTTTTTGTCAAAAGACCTTTT / i2FU / rArArU / i2FU / / i2FU / / i2FC / / i2FU / / i2FA / / i2FC / rU / i2FC / / i2FU / / i2FU / rG / i2FU / / i2FA / / i2FG / / i2FA / rUx (SEQ ID NO: 114); wherein x comprises or consists of the targeting domain, and wherein the 3’ nucleotide of the targeting domain comprises a 2’ O-methyl modification and is linked to an adjacent nucleotide by a phosphorothioate linkage.

[0066] In certain embodiments, the gRNA molecule comprises the following sequence: / 5InvdT / ATGTGTTTTTGTCAAAAGACCTTTTrUrArArUrUrUrCrUrArCrU rCrUrUrGrUrArGrArUx / 3InvdT / (SEQ ID NO: 115); wherein x comprises or consists of the targeting domain.

[0067] In certain embodiments, the gRNA molecule comprises the following sequence: / 5InvdT / ATGTGTTTTTGTCAAAAGACCTTTT / i2FU / rArArU / i2FU / / i2FU / r CrU / i2FA / / i2FC / rU / i2FC / / i2FU / / i2FU / rG / i2FU / / i2FA / / i2FG / / i2FA / rUx / 3InvdT (SEQ ID NO: 116); wherein x comprises or consists of the targeting domain.

[0068] In certain embodiments, the gRNA molecule comprises the following sequence: / 5InvdT / ATGTGTTTTTGTCAAAAGACCTTTT / i2FU / rArArU / i2FU / / i2FU / rCrU / i2 FA / / i2FC / rU / i2FC / / i2FU / / i2FU / rG / i2FU / / i2FA / / i2FG / / i2FA / rUx (SEQ ID NO: 441); wherein x comprises or consists of the targeting domain. In certain embodiments, the gRNA molecule comprises the following sequence: / i2FU / rArArU / i2FU / / i2FU / rCrU / i2FA / / i2FC / rU / i2FC / / i2FU / / i2FU / rG / i2FU / / i2FA / / i2 FG / / i2FA / rUx / 3InvdT / (SEQ ID NO: 442); wherein x comprises or consists of the targeting domain. In certain embodiments, the gRNA molecule comprises the following sequence: / i2FU / rArArU / i2FU / / i2FU / rCrU / i2FA / / i2FC / rU / i2FC / / i2FU / / i2FU / rG / i2FU / / i2FA / / i2 FG / / i2FA / rUx (SEQ ID NO: 443); wherein x comprises or consists of the targeting domain,

[0069] In certain embodiments, the gRNA molecule comprises the following sequence: mA*TGTGTTTTTGTCAAAAGACCTTTT / i2FU / rArArU / i2FU / / i2FU / rCrU / i 2FA / / i2FC / rU / i2FC / / i2FU / / i2FU / rG / i2FU / / i2FA / / i2FG / / i2FA / rUx (SEQ ID NO: 117); wherein x comprises or consists of the targeting domain, and w herein the 3 ’ nucleotide of the targeting domain comprises a 2’ O-methyl modification and is linked to an adjacent nucleotide by a phosphorothioate linkage.

[0070] In certain embodiments, the gRNA molecule comprises the following sequence: / 5InvdT / ATGTGTTTTTGTCAAAAGACCTTTTrUrArArU / i2FU / / i2FU / / i2FC / / i2FU / / i2FA / / i2FC / rU / i2FC / / i2FU / / i2FU / rG / i2FU / / i2FA / / i2FG / / i2FA / rUx / 3InvdT / (SEQ ID NO: 444); wherein x comprises or consists of the targeting domain. In certain embodiments, the gRNA molecule comprises the following sequence: / 5InvdT / ATGTGTTTTTGTCAAAAGACCTTTTrUrArArU / i2FU / / i2FU / / i2FC / / i2FU / / i2FA / / i2FC / rU / i2FC / / i2FU / / i2FU / rG / i2FU / / i2FA / / i2FG / / i2FA / rUx (SEQ ID NO:

[0071] 445); wherein x comprises or consists of the targeting domain. In certain embodiments, the gRNA molecule comprises the following sequence: / 5InvdT / ATGTGTTTTTGTCAAAAGACCTTTTrUrArArU / i2FU / / i2FU / rCrU / i2FA / / i2FC / rU / i2FC / / i2FU / / i2FU / rG / i2FU / / i2FA / / i2FG / / i2FA / rUx / 3InvdT / (SEQ ID NO:

[0072] 446); wherein x comprises or consists of the targeting domain. In certain embodiments, the gRNA molecule comprises the following sequence: / 5InvdT / ATGTGTTTTTGTCAAAAGACCTTTTrUrArArU / i2FU / / i2FU / rCrU / i2FA / / i2FC / rU / i2FC / / i2FU / / i2FU / rG / i2FU / / i2FA / / i2FG / / i2FA / rUx (SEQ ID NO: 447); wherein x comprises or consists of the targeting domain.

[0073] In certain embodiments, the gene of interest is expressed in a liver cell (e.g., a hepatocyte) of a subject.

[0074] In certain embodiments, the liver-expressed gene is LPA, ANGPTL3, PCSK9, LDLR, APOC2, APOC3. APOB. MTP, ANGPTL4, ANGPTL8, APOA5, ApoB, APOE, IDOL, NPC1L1, ASGR1, TM6SF2, GALNT2, LPL, MLXIPL. SORT1, TRIBI, MARC I, ABCG5, ABCG8, PNPLA3, TM6SF2, HFE, GCKR, HMOX-1, IIGT1A1, STAP1, LDLRAP1, LMF-1, GP1HBP1, CYP27A1, LIPA or ATP7B.

[0075] In certain embodiments, the gene of interest is expressed in a kidney cell (e.g., a renal epithelial cell) of a subject.

[0076] In certain embodiments, the gene of interest is expressed in a stem cell (e.g.. a CD34+hematopoietic stem and progenitor cell) of a subject.

[0077] In certain embodiments, the gene of interest is expressed in an ocular cell (e.g., a trabecular mesh work cell) of a subject. In certain embodiments, the gene of interest isMY OC.

[0078] In certain embodiments, the liver-expressed gene is involved in regulating lipid and / or cholesterol metabolism. In certain embodiments, decreasing the expression of the liver-expressed gene decreases the levels of circulating LDL. In certain embodiments, decreasing the expression of the liver-expressed gene decreases the levels of circulating Lp(a). In certain embodiments, the liver-expressed gene is LPA, ANGPTL3, PCSK9, LDLR, APOC2, APOC3, APOB, MTP, ANGPTL4, ANGPTL8, APOA5, ApoB, APOE, IDOL, NPC1L1, ASGR1, TM6SF2, GALNT2, LPL, MLXIPL, SORT!, TRIBI, MARC1, ABCG5, ABCG8, PNPLA3, TM6SF2, HEE, HMOX-1, UGT1A1, STAP1, LDLRAP1, IMF-1, GP1HBP1, CYP27A1, LIPA or A TP7B

[0079] BRIEF DESCRIPTION OF THE DRAWINGS

[0080] The accompanying drawings are intended to provide illustrative, and schematic rather than comprehensive, examples of certain aspects and embodiments of the present disclosure. The drawings are not intended to be limiting or binding to any particular theory or model. Without limiting the foregoing, nucleic acids and polypeptides can be depicted as linear sequences, or as schematic two- or three-dimensional structures; these depictions are intended to be illustrative rather than limiting or binding to any particular model or theory’ regarding their structure.

[0081] FIGs. 1A-1E depict the results of an RNP nucleofection screen for AsCasl2a gRNAs targeting LPA, ANGPTL3 and PCSK9. FIG. 1A shows the results of the LPA screen in T cells. FIGs. IB- IE show the results of the LPA, ANGPTL3, and PCSK9 screen in HepG2 cells. Briefly, gRNAs were complexed with the appropriate nuclease and electroporated into primary human T cells at 8 mM (FIG. 1 A) or HepG2 cells, a human hepatocellular carcinoma cell line, at 2 mM (FIGs. 1B-1E). Indel Fraction (the percent of reads with an insertion or deletion mutation) w as determined by NGS. Tw o w ells of cells for each were analyzed, and lines depict the mean indel fraction (equivalent to % editing). For FIGs. 1A-1B, AsCasl2a-MHF / TTTV refers to RNP complexes incorporating WT AsCasl2a, which recognizes a TTTV PAM, MHFRR / TYCV and MHFRR / CCCC refer to RNP complexes incorporating an AsCasl2a- MHFRR variant that recognizes the alternative PAM sequences TYCV and CCCC, and MHFRVR / TATV refers to RNP complexes incorporating an AsCasl2a-MHFRVR variant which recognizes the alternative PAM sequence TATV.

[0082] FIGs. 2A-2C depict exemplar}’ results of lipid nanoparticle (LNP)-mediated LPA editing. FIG. 2A depicts LPA editing in primary’ human hepatocytes (PHHs) treated with LNPs containing AsCasl2amRNA and LPA -targeting gRNA. FIG. 2B depicts LPA editing inHep3B cells. FIG. 2C depicts LPA editing in HepG2 cells. FIG. 2D depicts LPA editing and protein knockdown in PHHs isolated from a single donor.

[0083] FIG. 3 illustrates examples of chemical modifications used for gRNAs including 2’Fluoro modification (or 2'Fluorine modification), phosphorothioate modification, 2’0 Methyl modification, 5’-5’ inverted dT, and 3’-3’ inverted dT. FIG. 4 depicts the evaluation of additional gRNAs targeting LPA in PHHs using specified LNP delivery format, including certain gRNA modifications (see Table 19).

[0084] FIGs. 5A-5I depict exemplary dose response editing analysis for liver cells treated with LNPs containing AsCasl2a mRNA and either LPA -targeting or MYOC-targeting gRNA with various chemical modifications. FIG. 5A depicts the results of LPA editing efficiency with the indicated gRNA in Hep3B cells as a function of AsCasl2a mRNA concentrations. All gRNAs include SEQ ID NO: 39 as the targeting domain. FIG. 5B depicts the results of LPA editing efficiency with the indicated gRNA in PHHs as a function of AsCasl 2a mRNA concentrations. All gRNAs include SEQ ID NO: 39 as the targeting domain. FIG. 5C shows a comparison between guide 589 having an ‘‘aggressive” hairpin 2’F pattern (“Agg”) and guide 614 having a “conservative” hairpin 2'F pattern (“Con”) for LPA editing efficiency in PHHs. FIG. 5D shows a comparison between guide 591 and guide 616 for LPA editing efficiency in PHHs. FIG. 5E shows a comparison between guide 615 and guide 599 for LPA editing efficiency in PHHs. FIG. 5F shows a comparison between guide 617 and guide 600 for LPA editing efficiency in PHHs. FIG. 5G shows a second comparison experiment between guide 589 and guide 614 for LPA editing efficiency in PHHs. FIG. 5H shows MYOC editing efficiency in Hep3B cells using a gRNA of SEQ ID NO: 107. FIG. 51 shows MYOC editing efficiency in PHHs using a gRNA of SEQ ID NO: 107.

[0085] FIG. 6 depicts the results of LNP mediated LPA editing in PHHs isolated from multiple donors.

[0086] FIGs. 7A-7C depict exemplary results for in vivo MYOC editing using LNPs in the liver of humanized myocilin (MYOC) mouse. FIG 7A shows the experimental design. Briefly, a dose curve using a guide containing a 5’ extension and idT and 2’F modifications was performed. For FIG. 7B, guide 593 was formulated into LNPs with engineered AsCasl2a- MHF mRNA using an ALC-0315-based LNP formulation (50% ALC-0315, 38.5% cholesterol, 10% DSPC, 1.5% DMG-PEG2k), and administered at the specified dose to mice by IV tail vein injection. For FIG. 7C, gRNAs containing the specified modifications were similarly formulated and administered at a dose of 0. 1 mg / kg (see Table 22 for gRNA sequences).

[0087] FIG. 8 indicates the percentage of indels introduced into the MYOC gene in primary human trabecular meshwork (TM) cells resulting from transfection with LNPs encapsulating AsCasl2a mRNA plus gRNAs comprising different modifications (see Table 25 for gRNA sequences).

[0088] FIGs. 9A and 9B depict MY 01 -HOM mouse in vivo data resulting from administration of LNPs encapsulating AsCasl2a mRNA plus gRNAs comprising different modifications (See Table 25 for gRNA sequences). FIG. 9A indicates the percentage of myocilin mRNA remaining after editing as determined by RT-ddPCR. FIG. 9B indicates the percentage of indels introduced into the MYOC gene determined by Ill-Seq.

[0089] FIGs. 10A and 10B depict the effect of gRNA chemical modifications on MYOC editing potency in vitro in HEK293T cells, where the gRNA targets a region (SEQ ID NO: 137) of the MYOC gene. FIG. 10A depicts the effect of single modifications to the gRNA on the editing efficiency of the MYOC gene. FIG. 10B depicts the effect of both single and dual modifications to the gRNA on the editing efficiency of the MYOC gene.

[0090] FIGs. 11A and 11B depict the effect of gRNA chemical modifications on MYOC editing potency in vitro and in vivo in TM cells, where the gRNA targets a region (SEQ ID NO: 123) of the MYOC gene that is different from that targeted for the results shown in FIGs. 10A-10B. FIG. 11A depicts the effect of the indicated gRNA chemical modifications on the editing efficiency of the MYOC gene in vitro. FIG. 11B depicts the results of in vivo editing using gRNAs comprising chemical modifications #3, #4 and #5 as described herein.

[0091] FIGs. 12A-12G depict the effect of gRNA chemical modifications on the editing efficiency of the MYOC gene in various cell types. FIG. 12A depicts the effect of gRNA chemical modifications on the editing efficiency in PHHs. FIG. 12B depicts the effect of gRNA chemical modifications on the editing efficiency in TM cells. FIG. 12C depicts the effect of gRNA chemical modifications on the editing efficiency in primary CD34+ cells. FIG. 12D depicts the effect of gRNA chemical modifications on the editing efficiency in primary renal epithelial cells. FIG. 12E depicts the effect of gRNA chemical modifications on the editing efficiency in pancreatic ductal cells. FIG. 12F depicts the effect of gRNA chemical modifications on the editing efficiency in HepG2 cells. FIG. 12G depicts the effect of gRNA chemical modifications on the editing efficiency in Hep3B cells.

[0092] FIGs. 13A-13D depict the results of an in vitro binding affinity assay designed to study the effect of chemical modifications of the MYOC-targ eting gRNA on its ability to bind to AsCasl2a nuclease. FIG. 13A illustrates the assay procedure used for this study. FIG. 13B depicts the percent (%) bound labeled gRNA as a function of unlabeled test gRNA concentration for a gRNA targeting MYOC. FIG. 13C depicts % bound labeled gRNA as a function of unlabeled test gRNA concentration for a gRNA targeting LPA. FIG. 13D depicts % bound labeled gRNA as a function of unlabeled test gRNA concentration for a different gRNA targeting MYOC.

[0093] FIGs. 14A-14C depict ANGPTL3 editing and protein knockdown in PHHs isolated from multiple donors treated with LNPs containing AsCas 12a mRNA and ANGPTL3-targeting gRNA of SEQ ID NO: 388. FIGs. 14D-14E depict LPA, ANGPTL3 andPCSK9 editing in PHH and HepG2 cells treated with LNPs containing AsCasl2A mRNA and LPA-, ANGPTL3-, or CS 9-targeting gRNA of SEQ ID NO: 98 (LPA 589), SEQ ID NO: 388 (ANGPTL3 496), SEQ ID NO: 389 (ANGPTL3 497), SEQ ID NO: 390 (PCSK9 498), or SEQ ID NO: 391 (PCSK9 499).

[0094] FIG. 15 depicts exemplary results for in vivo LPA editing using LNPs comprising the ionizable lipid ALC-0315 in the liver of humanized LPA mice.

[0095] FIGs. 16A-16B depict exemplary results for in vivo LPA editing using LNPs comprising the ionizable lipid ALC-0315 in the liver of humanized LPA mice, measured by Jess Western assay. Briefly, the assay was optimized by characterizing performance of several different Apo(a) antibodies, ability to detect Apo(a) at different concentrations, and specificity for human Apo(a) in mice. FIG. 16A compares the Jess signal of purified Lp(a) to neat WT mouse serum or WT mouse serum spiked with Lp(a). FIG. 16B depicts serum Apo(a) in predose and post-dose mice.

[0096] FIG. 17 depicts exemplary results for in vivo LPA editing and Apo(a) knockdown using LNPs comprising the ionizable lipid ALC-0315 in the liver of humanized LPA mice.

[0097] FIG. 18 depicts exemplary results for in vivo ANGPTL3 editing using LNPs in the liver of wild type C57B1 / 6 (WT) mice. A surrogate guide targeting mouse ANGPTL3 was formulated into LNPs with engineered AsCasl2a mRNA using an ALC-0315-based LNP formulation (50% ALC-0315, 38.5% cholesterol. 10% DSPC, 1.5% DMG-PEG2k), and administered at the specified dose to mice by IV tail vein injection.

[0098] FIGs. 19A-19B illustrate exemplary results for in vivo mouse ANGPTL3 protein knockdown in WT mice treated with LNPs encapsulating AsCasl2a mRNA plus surrogate gRNA targeting mouse ANGPTL3. FIG. 19A depicts the serum concentration of mouse ANGPTL3 treated WT mice at different LNP doses. FIG. 19B depicts the data of FIG. 19A as percent knockdown normalized to the vehicle control.

[0099] FIG. 20 depicts exemplary' results for in vivo ANGPTL3 editing using LNPs in the liver of humanized ANGPTL3 transgenic mice. A guide targeting human ANGPTL3 was formulated into LNPs with engineered AsCasl2a mRNA using an ALC-0315-based LNP formulation (50% ALC-0315, 38.5% cholesterol, 10% DSPC, 1.5% DMG-PEG2k), and administered at the specified dose to mice by IV tail vein injection.

[0100] FIGs. 21A-21B depict exemplary results for human ANGPTL3 protein detection in humanized ANGPTL3 transgenic mice. FIG. 21A depicts human and mouse ANGPTL3 protein in plasma of WT (+ / +) and humanized ANGPTL3 transgenic mice (H / H). FIG. 21B depicts human ANGPTL3 protein in plasma of pre-dose humanized ANGPTL3 transgenic mice, quantified by a low limit of quantification (LoQ) ELISA.

[0101] FIGs. 22A-22B depict exemplary results for in vivo ANGPTL3 editing using LNPs in the liver of humanized ANGPTL3 transgenic mice. A guide targeting human ANGPTL3 was formulated into LNPs with engineered AsCasl2a mRNA using an ALC-0315-based LNP formulation (50% ALC-0315, 38.5% cholesterol, 10% DSPC, 1.5% DMG-PEG2k), and administered at the specified dose to mice by IV tail vein injection. FIG. 22A depicts indel generation in human ANGPTL3 and knockdown of human ANGPTL3 protein in liver tissue following LNP administration. FIG. 22B depicts human ANGPTL3 protein concentration in liver tissue of pre-dose and terminal mice.

[0102] DETAILED DESCRIPTION

[0103] The presently disclosed subject matter relates to RNA-guided nuclease-related, e.g.. CRISPR / Cas-related, genome editing systems, compositions, delivery vehicles, and methods for targeting a gene of interest, editing a, or modulating expression of a gene of interest, and applications thereof. The presently disclosed subject matter also provides genome editing systems, compositions, vectors, and methods for editing cells using CRISPR / Cas-related components to edit a target gene of interest. The presently disclosed subject matter also provides lipid nanoparticle (LNP) facilitated gene delivery of genome editing systems and methods for editing cells using CRISPR / Cas-related components delivered via LNP to edit a target liver-expressed gene (e.g. , LPA, ANGPTL3, PCSK9. LD L R) Some aspects of the present disclosure provide pharmaceutical compositions, cells, cell populations, methods, strategies, and treatment modalities that are useful in the context of treating and / or managing a metabolic disease, e.g., hyperlipidemia or hypercholesterolemia.

[0104] The subject matter of the present disclosure is described with reference to the Figures. It should be understood that numerous specific details, relationships, and methods are set forth in this Detailed Description, Examples, and accompanying Figures to provide a more complete understanding of the subject matter disclosed herein. For purposes of clarity of disclosure and not by way of limitation, the Detailed Description is divided into the following subsections:

[0105] Definitions and Abbreviations

[0106] Genome editing systems

[0107] Guide RNA (gRNA) molecules

[0108] Guide RNA design

[0109] RNA-guided nucleases Genome editing strategies

[0110] Implementation of genome editing systems: delivery, formulations, and routes of administration

[0111] Examples

[0112] 1. Definitions and Abbreviations

[0113] Unless otherwise specified, each of the following terms has the meaning associated with it in this section.

[0114] The indefinite articles “a” and '‘an” refer to at least one of the associated noun and are used interchangeably with the terms “at least one” and “one or more.” For example, “a module” means at least one module, or one or more modules.

[0115] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 3 or more than 3 standard deviations, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value.

[0116] The conjunctions “or” and “and / or” are used interchangeably as non-exclusive disjunctions.

[0117] “Domain” is used to describe a segment of a protein or nucleic acid. Unless otherwise indicated, a domain is not required to have any specific functional properly .

[0118] An “indel” is an insertion and / or deletion in a nucleic acid sequence. An indel can be the product of the repair of a DNA double strand break, such as a double strand break formed by a genome editing system of the present disclosure. An indel is most commonly formed when a break is repaired by an “error prone” repair pathway such as the NHEJ pathway described below.

[0119] “Gene conversion” refers to the alteration of a DNA sequence by incorporation of an endogenous homologous sequence (e.g.. a homologous sequence within a gene array). “Gene correction” refers to the alteration of a DNA sequence by incorporation of an exogenous homologous sequence, such as an exogenous single-or double stranded donor template DNA. Gene conversion and gene correction are products of the repair of DNA double-strand breaks by HDR pathways such as those described below. Indels, gene conversion, gene correction, and other genome editing outcomes are ty pically assessed by sequencing (most commonly by "next-gen” or "sequencing-by-synthesis” methods, though Sanger sequencing can still be used) and are quantified by the relative frequency of numerical changes (e.g, ±1, +2 or more bases) at a site of interest among all sequencing reads. DNA samples for sequencing can be prepared by a variety' of methods known in the art and can involve the amplification of sites of interest by polymerase chain reaction (PCR). the capture of DNA ends generated by double strand breaks, as in the GUIDE-seq process described in Tsai et al. (Nat. Biotechnol. 34(5): 483 (2016), incorporated by reference herein) or by other means well know n in the art. Genome editing outcomes can also be assessed by in situ hybridization methods such as the FiberComb™ system commercialized by Genomic Vision (Bagneux, France), and by any other suitable methods known in the art. Alt-HDR,” ‘'alternative homology -directed repair,” or ‘'alternative HDR” are used interchangeably to refer to the process of repairing DNA damage using a homologous nucleic acid (e.g., an endogenous homologous sequence, e.g., a sister chromatid, or an exogenous nucleic acid, e.g. , a template nucleic acid). Alt-HDR is distinct from canonical HDR in that the process utilizes different pathways from canonical HDR, and can be inhibited by the canonical HDR mediators, RAD51 and BRCA2. Alt-HDR is also distinguished by the involvement of a single-stranded or nicked homologous nucleic acid template, whereas canonical HDR generally involves a double-stranded homologous template.

[0120] “Canonical HDR,” “canonical homology-directed repair” or “cHDR” refer to the process of repairing DNA damage using a homologous nucleic acid (e.g, an endogenous homologous sequence, e.g, a sister chromatid, or an exogenous nucleic acid, e.g., a template nucleic acid). Canonical HDR ty pically acts when there has been significant resection at the double strand break, forming at least one single stranded portion of DNA. In a normal cell, cHDR typically involves a series of steps such as recognition of the break, stabilization of the break, resection, stabilization of single stranded DNA, formation of a DNA crossover intermediate, resolution of the crossover intermediate, and ligation. The process requires RAD51 and BRC A2, and the homologous nucleic acid is typically double stranded.

[0121] Unless indicated otherwise, the term “HDR” as used herein encompasses both canonical HDR and alt-HDR.

[0122] “Non-homologous end joining” or “NHEJ” refers to ligation mediated repair and / or non-template mediated repair including canonical NHEJ (cNHEJ) and alternative NHEJ (altNHEJ), which in turn includes microhomology-mediated end joining (MMEJ), single- strand annealing (SSA), and synthesis-dependent microhomology -mediated end joining (SD- MMEJ). ‘Replacement” or “replaced,” when used with reference to a modification of a molecule (e.g., a nucleic acid or protein), does not require a process limitation but merely indicates that the replacement entity is present.

[0123] “Knock-out” or “knockout” refers to an inactivating mutation in a target gene, wherein the product of the target gene comprises a loss of function.

[0124] “Gene product” refers to biochemical products resulting from the expression of the gene and includes the RNA or protein that is encoded by the gene.

[0125] “On-target site” refers to the exact genomic sequence or locus within the gene of interest for which the guide RNA / RNA-guided nuclease was designed to target. “Off-target site” refers to a genomic sequence or locus that is not the on-target site and may be (or is found to be) edited by the RNA guided nucleases.

[0126] As used herein, the term “hairpin” or “hairpin region” refers to a nucleic acid secondary structure comprising a stem-loop structure. A guide-RNA of the present disclosure may comprise one or more hairpins, or hairpin regions, that are not part of its targeting domain.

[0127] As used herein, the term “pseudoknot” refers to a nucleic acid secondary structure comprising at least two stem-loop structures in which half of one stem is intercalated between the two halves of another stem. Exemplary pseudoknot sequences are bolded in the two gRNA sequences shown below; rUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrGrGrCrCrUrGrArGrArUrGrCrC rArGrCrUrGrUrCrCrUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArU [SEQ ID NO: 257]; mU*rArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrGrGrCrCrUrGrArGrArUrGrC rCrArGrCrUrGrUrCrCrUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrA*mU [SEQ ID NO: 258],

[0128] As used herein, the term “locked nucleic acid” or “LNA” refers to a modified RNA nucleotide in which the ribose moiety is modified with a bridge connecting the 2’ oxygen and 4’ carbon. An exemplary nucleic acid including a LNA is shown below, where the “+” indicates a locked nucleotide;

[0129] / 5IdT / +A+T+G+T+G+T+T+T+T+T+G+T+C+A+A+A+A+G+A+C+C+T+T+T+TrUr ArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrCrArArCrCrUrCrCrUrGrGrCrCrArGrArUr UrCrUrC / 3InvdT / [SEQ ID NO: 275], “Subject’' means a human or non-human animal. A human subject can be any age (e.g., an infant, child, young adult, or adult), and can suffer from a disease, or can be in need of alteration of a gene. Alternatively, the subject can be an animal, which term includes, but is not limited to, mammals, birds, fish, reptiles, amphibians, and more particularly non-human primates (NHP), rodents (such as mice, rats, hamsters, etc.), rabbits, guinea pigs, dogs, cats, and so on. In certain embodiments of this disclosure, the subject is livestock, e.g., a cow, a horse, a sheep, or a goat. In certain embodiments, the subject is poultry.

[0130] As used herein a “therapeutically effective amount” refers to the amount of a cell and / or composition that when administered to a subject for treating a disease, is sufficient to beneficially affect such treatment for the disease.

[0131] “Treat,” “treating,” and “treatment” mean the treatment of a disease in a subject (e.g. , a human subject), including one or more of inhibiting the disease, z.e., arresting or preventing its development or progression; relieving the disease, i.e., causing regression of the disease state; relieving one or more symptoms of the disease; and curing the disease.

[0132] “Prevent,” “preventing,” and “prevention” refer to the prevention of a disease in a mammal, e.g., in a human, including (a) avoiding or precluding the disease; (b) affecting the predisposition toward the disease; or (c) preventing or delaying the onset of at least one symptom of the disease.

[0133] A “Kit"’ refers to any collection of two or more components that together constitute a functional unit that can be employed for a specific purpose. By way of illustration (and not limitation), one kit according to this disclosure can include a guide RNA complexed or able to complex with an RNA-guided nuclease, and accompanied by (e.g., suspended in, or suspendable in) a pharmaceutically acceptable carrier. The kit can be used to introduce the complex into, for example, a cell or a subject, for the purpose of causing a desired genomic alteration in such cell or subject. The components of a kit can be packaged together, or they can be separately packaged. Kits according to this disclosure also optionally include directions for use (DFU) that describe the use of the kit e.g., according to a method of this disclosure. The DFU can be physically packaged with the kit. or it can be made available to a user of the kit, for instance by electronic means.

[0134] The terms “polynucleotide”, “nucleotide sequence”, “nucleic acid”, “nucleic acid molecule”, “nucleic acid sequence”, and “oligonucleotide” mean any chain of two or more nucleotide bases (also called nucleotides). The polynucleotides, nucleotide sequences, nucleic acids etc. can be chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded. They can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, its hybridization parameters, etc. A nucleotide sequence typically carries genetic information, including, but not limited to, the information used by cellular machinery to make proteins and enzymes. These terms include double- or single-stranded genomic DNA, RNA, any synthetic and genetically manipulated polynucleotide, and both sense and antisense polynucleotides. These terms also include nucleic acids containing modified bases.

[0135] Conventional IUPAC notation is used in nucleotide sequences presented herein, as shown in Table 1, below (see also Cornish-Bowden A, Nucleic Acids Res. 1985 May 10; 13(9):3021-30, incorporated by reference herein). It should be noted, however, that “T” denotes “Thymine or Uracil” in those instances where a sequence can be encoded by either DNA or RNA, for example in a gRNA. for example in a gRNA targeting domain.

[0136] Table 1: IUPAC nucleic acid notation

[0137] The terms “protein,” “peptide” and “polypeptide” are used interchangeably to refer to a sequential chain of amino acids linked together via peptide bonds. The terms include individual proteins, groups or complexes of proteins that associate together, as well as fragments or portions, variants, derivatives and analogs of such proteins. Peptide sequences are presented herein using conventional notation, beginning with the amino or N-terminus on the left, and proceeding to the carboxyl or C-terminus on the right. Standard one-letter or three- letter abbreviations can be used. The term “variant’' refers to an entity such as a polypeptide, polynucleotide or small molecule that shows significant structural identity’ with a reference entity but differs structurally from the reference entity in the presence or level of one or more chemical moieties as compared with the reference entity’. In many’ embodiments, a variant also differs functionally from its reference entity . In general, whether a particular entity’ is properly considered to be a “variant’' of a reference entity is based on its degree of structural identity with the reference entity.

[0138] As used herein, the term “promoter” refers to a region (I.e., a DNA sequence) of a genome that initiates the transcription of a gene.

[0139] The term “endogenous,” as used as used herein in the context of nucleic acids (e.g., genes, protein-encoding genomic regions, promoters) or proteins, refers to a native nucleic acid or protein in its natural location, e.g., within the genome of a cell. In contrast, the term “exogenous,” as used herein in the context of nucleic acids, e.g., expression constructs, cDNAs, indels, and nucleic acid vectors, or proteins, refers to nucleic acids or proteins that have artificially been introduced into the cell. For example, an exogenous nucleic acid may be introduced into the genome of a cell using, for example, gene-editing or genetic engineering techniques, e.g., CRISPR-based editing techniques.

[0140] The terms “RNA-guided nuclease” and “RNA-guided nuclease molecule” are used interchangeably herein. In some embodiments, the RNA-guided nuclease is an RNA-guided DNA endonuclease enzyme. In some embodiments, the RNA-guided nuclease is a CRISPR nuclease. Non-limiting examples of RNA-guided nucleases are listed in Table 2 below, and the methods and compositions disclosed herein can use any combination of RNA-guided nucleases disclosed herein, or known to those of ordinary skill in the art. Those of ordinary skill in the art will be aware of additional nucleases and nuclease variants suitable for use in the context of the present disclosure, and it will be understood that the present disclosure is not limited in this respect.

[0141] Table 2: RNA-Guided Nucleases

[0142] Additional suitable RNA-guided nucleases, e.g., Cas9 and Casl2 nucleases, will be apparent to the skilled artisan in view of the present disclosure, and the disclosure is not limited by the exemplary' suitable nucleases provided herein. In some embodiment, a suitable nuclease is a Cas9 or Casl2a (Cpfl) nuclease. In some embodiments, the disclosure also embraces nuclease variants, e.g., Cas9 or Casl2a nuclease variants. A nuclease variant refers to a nuclease comprising an amino acid sequence characterized by one or more amino acid substitutions, deletions, or additions as compared to the wild type (WT) amino acid sequence of the nuclease. Suitable nucleases and nuclease variants may also comprise purification tags (e.g., polyhistidine tags) and signaling peptides, e.g., comprising or consisting of a nuclear localization signal sequence (NLS). Some non-limiting examples of suitable nucleases and nuclease variants are described in more detail elsewhere herein, and also comprise those described in PCT application PCT / US2019 / 22374, filed March 14, 2019, and entitled “Systems and Methods for the Treatment of Hemoglobinopathies f the entire contents of which are incorporated herein by reference.

[0143] In some embodiments, the RNA-guided nuclease is an Acidaminococcus sp. Casl2a (Cpfl) variant (also known as AsCasl2a or AsCpfl variant). Suitable Casl2a nuclease variants, including suitable AsCasl2a variants will be known or apparent to those of ordinary skill in the art based on the present disclosure, and include, but are not limited to, the AsCasl2a variants disclosed herein or otherwise known in the art. For example, in some embodiments, the RNA-guided nuclease is an Acidaminococcus sp. Casl2a RR variant (AsCasl2a-RR). In certain embodiments, the RNA-guided nuclease is a Casl2a RVR variant. These and other variants are described in PCT patent application PCT / US2017 / 028420 and Guo et al., Nat Biotechnol. 2017 Aug; 35(8): 789-792, each of which is incorporated by reference herein for all purposes. Additionally, or alternatively, suitable Casl2a variants include those having an M537R substitution, an H800A substitution, and / or an F870L substitution, or any combination thereof (numbering scheme according to As Casl2a wild-ty pe sequence).

[0144] In certain embodiments, the RNA-guided nucleases of the present disclosure can comprise a DNA modifying enzyme for targeted nucleotide alteration, commonly referred to as a "base editor.” In certain embodiments, the base editor is a cytosine base editor. In certain embodiments, the base editor is an adenosine base editor.

[0145] In certain embodiments, the RNA-guided nucleases of the present disclosure can comprise a DNA modifying enzyme fused to a reverse transcriptase for targeted nucleotide insertion, deletion, or substitution, commonly referred to as a “prime editor.”

[0146] 2. Genome editing systems

[0147] Various genome editing systems known in the art can be used for the methods disclosed herein. Non-limiting examples of genome editing systems that can be used with the presently disclosed subject matter include, but are not limited to CRISPR systems, zinc-finger nuclease (ZFN) systems, transcription activator-like effector nuclease (TALEN) systems, meganuclease (MN) systems, MegaTAL systems, other targeted endonuclease systems, and other chimeric endonuclease systems.

[0148] In certain embodiments, the genome editing system has RNA-guided DNA editing activity. In certain embodiments, the genome editing system includes at least two components adapted from naturally occurring CRISPR systems: a guide RNA (gRNA) and an RNA-guided nuclease. These two components form a complex that is capable of associating with a specific nucleic acid sequence and optionally editing the DNA in or around that nucleic acid sequence, for instance by making one or more of a single-strand break (an SSB or nick), a double-strand break (a DSB) and / or a point mutation.

[0149] Naturally occurring CRISPR systems are organized evolutionarily into two classes and five types (Makarova et al. Nat Rev Microbiol. 2011 Jun; 9(6): 467-477 (Makarova), incorporated by reference herein), and while genome editing systems of the present disclosure can adapt components of any type or class of naturally occurring CRISPR system, the embodiments presented herein are generally adapted from Class 2, and type II or V CRISPR systems. Class 2 systems, which encompass types II and V, are characterized by relatively large, multidomain RNA-guided nuclease proteins (e.g., Cas9 or Casl2a) and one or more guide RNAs (e.g., a crRNA and, optionally, a tracrRNA) that form ribonucleoprotein (RNP) complexes that associate with (z.e., target) and cleave specific loci complementary to a targeting (or spacer) sequence of the crRNA. Genome editing systems according to the present disclosure similarly target and optionally edit cellular DNA sequences but differ significantly from CRISPR systems occurring in nature. For example, the unimolecular guide RNAs described herein do not occur in nature, and both guide RNAs and RNA-guided nucleases according to this disclosure can incorporate any number of non-naturally occurring modifications.

[0150] Genome editing system disclosed herein can be delivered into cell by electroporation. Other non-viral approaches can also be employed for genome editing of target cells disclosed herein. For example, a nucleic acid molecule can be introduced into cells / subjects by administering the nucleic acid in the presence of lipofection (Feigner et al., Proc. Natl. Acad. Sci. U.S.A. 84:7413. 1987; Ono et al., Neuroscience Letters 17:259. 1990; Brigham et al., Am. J. Med. Sci. 298:278, 1989; Staubinger et al., Methods in Enzymology 101 :512, 1983), asialoorosomucoid-polylysine conjugation (Wu et al., Journal of Biological Chemistry 263: 14621, 1988; Wu et al., Journal of Biological Chemistry 264: 16985. 1989), or by microinjection under surgical conditions (Wolff et al.. Science 247: 1465, 1990). Other non-viral means for gene transfer include transfection in vitro using calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Lipid nanoparticles (LNPs) or liposomes are also contemplated for delivery of nucleic acid molecules into a cell. In some embodiments, the genome editing systems disclosed herein are delivered in vivo to a subject by administration of lipid nanoparticles (LNPs) containing one or more components of the genome editing system.

[0151] Genome editing system disclosed herein can be delivered into subjects or cells using viral vectors, e.g., retroviral vectors, gamma-retroviral vectors, or lentiviral vectors. Combinations of a retroviral vector and an appropriate packaging line are suitable, where the capsid proteins will be functional for infecting human cells. Various amphotropic virusproducing cell lines are known, including, but not limited to, PA12 (Miller, et al. (1985) Mol. Cell. Biol. 5:431-437); PA317 (Miller, et al. (1986) Mol. Cell. Biol. 6:2895-2902); and CRIP (Danos, et al. (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464). Non-amphotropic particles are suitable too, e.g.. particles pseudotyped with VSVG, RD114 or GALV envelope and any other know n in the art. Possible methods of transduction also include direct co-culture of the cells with producer cells, e.g., by the method of Bregni, et al. (1992) Blood 80: 1418-1422, or culturing with viral supernatant alone or concentrated vector stocks with or without appropriate growth factors and polycations, e.g., by the method of Xu, et al. (1994) Exp. Hemat. 22:223- 230; and Hughes, et al. (1992) J. Clin. Invest. 89: 1817.

[0152] Genome editing systems can be implemented (e.g., administered or delivered to a cell or a subject) in a variety' of ways, and different implementations can be suitable for distinct applications. For instance, a genome editing system is implemented, in certain embodiments, as a protein / RNA complex (a ribonucleoprotein complex, or RNP complex), which can be included in a pharmaceutical composition that optionally includes a pharmaceutically acceptable carrier and / or an encapsulating agent, such as a lipid or polymer micro- or nanoparticle, micelle, liposome, etc. In certain embodiments, a genome editing system is implemented as one or more nucleic acids encoding the RNA-guided nuclease and / or guide RNA components described above (optionally with one or more additional components). In certain embodiments, the genome editing system is implemented as one or more vectors comprising such nucleic acids, for instance a viral vector such as an adeno-associated virus. In certain embodiments, the genome editing system is implemented as a combination of any of the foregoing. Additional or modified implementations that operate according to the principles set forth herein will be apparent to the skilled artisan and are within the scope of this disclosure.

[0153] It should be noted that the genome editing systems of the present disclosure can be targeted to a single specific nucleotide sequence or can be targeted to — and capable of editing in parallel — two or more specific nucleotide sequences through the use of two or more guide RNAs. The use of multiple gRNAs is referred to as '‘multiplexing” throughout this disclosure, and can be employed to target multiple, unrelated target sequences of interest, or to form multiple SSBs or DSBs within a single target domain and, in some cases, to generate specific edits within such target domain. For example. International Patent Publication No. WO 2015 / 138510 by Maeder et al. (Maeder), which is incorporated by reference herein, describes a genome editing system for correcting a point mutation (C.2991+1655A to G) in the human CEP290 gene that results in the creation of a cryptic splice site, which in turn reduces or eliminates the function of the gene. The genome editing system of Maeder utilizes two guide RNAs targeted to sequences on either side of (i.e., flanking) the point mutation, and forms DSBs that flank the mutation. This, in turn, promotes deletion of the intervening sequence, including the mutation, thereby eliminating the cryptic splice site, and restoring normal gene function.

[0154] Genome editing systems can, in some instances, form double strand breaks that are repaired by cellular DNA double-strand break mechanisms such as NHEJ or HDR. These mechanisms are described throughout the literature, for example by Davis & Maizels, PNAS, l l l(10):E924-932. March 11. 2014 (Davis) (describing Alt-HDR); Frit et al. DNA Repair 17(2014) 81-97 (Frit) (describing Alt-NHEJ); and lyama and Wilson III, DNA Repair (Amst.) 2013-Aug; 12(8): 620-636 (lyama) (describing canonical HDR and NHEJ pathways generally).

[0155] Where genome editing systems operate by forming DSBs, such systems optionally comprise one or more components that promote or facilitate a particular mode of double-strand break repair or a particular repair outcome. For instance, Cotta-Ramusino also describes genome editing systems in which a single stranded oligonucleotide "donor template” is added; the donor template is incorporated into a target region of cellular DNA that is cleaved by the genome editing system and can result in a change in the target sequence.

[0156] In certain embodiments, genome editing systems modify a target sequence, or modify expression of a gene in or near the target sequence, without causing double-strand breaks, e.g, by causing single-strand breaks or no cleavage ( / .e., no strand breaks). For example, a genome editing system can comprise an RNA-guided nuclease fused to a functional domain that acts on DNA. thereby modifying the target sequence or its expression. As one example, an RNA- guided nuclease can be connected to (e.g., fused to) a cytidine deaminase functional domain, and can operate by generating targeted C-to-A substitutions. An RNA-guided nuclease can also, for example, be connected to (e.g. fused to) an adenosine deaminase functional domain. Exemplary nuclease / deaminase fusions are described in Komor et al. Nature 533, 420-424 (19 May 2016) (“Komor”) and Kantor et al., Int. J. Mol. Sci. 21(17) 6240 (2020), which are hereby incorporated by reference in their entirety. Further non-limiting examples of suitable base editors, variants thereof, and strategies for preparing RNA-guided nucleases comprising the same are described in PCT applications: PCT / US2020 / 016664. filed February 4. 2020; PCT / US2020 / 0I8192, filed February 13, 2020; PCT / US2020 / 049975. field September 9, 2020; PCT / US2022 / 012054, filed January 11, 2022; and PCT / US2022 / 078655, filed October 25, 2022, the entire contents of each of which are incorporated herein by reference.

[0157] Alternatively, a genome editing system can utilize a cleavage-inactivated (i.e., a “dead”) nuclease, such as a dead Cas9 (dCas9), and can operate by forming stable complexes on one or more targeted regions of cellular DNA, thereby recruiting other functional domains and / or interfering with functions involving the targeted region(s) including, without limitation, mRNA transcription, chromatin remodeling, among others.

[0158] In certain embodiments, the RNA-guided nucleases of the present disclosure can comprise a polymerase domain (e.g., a reverse transcriptase domain). In certain embodiments, the RNA-guided nuclease may use a gRNA with a primer binding sequence and / or a template for the polymerase domain.

[0159] In certain embodiments, the RNA-guided nuclease may be a prime editor (PE), where the PE is an RNA-guided nuclease with nickase activity that is fused to a reverse transcriptase domain. In certain embodiments, the PE may use a prime editing gRNA (pegRNA), where the pegRNA is a gRNA with a primer binding sequence (PBS) and a donor template, e.g., added at one of the termini, e.g., the 3' end. In certain embodiments, aPE:pegRNA complex binds to the target DNA, and the nickase domain of the prime editor nicks only one strand, generating a flap. The PBS, located on the pegRNA, binds to the DNA flap and the edited RNA sequence is reverse transcribed using the reverse transcriptase domain of the prime editor. The edited strand is incorporated into the DNA at the end of the nicked flap, and the target DNA is repaired with the new reverse transcribed DNA. The original DNA segment is removed by a cellular endonuclease. Additional methods employing RNA-guided nucleases and polymerases for template mediated genome editing are described in PCT publications: WO 2020 / 191233, WO 2020 / 191248, WO 2021226558. WO2023283246, WO 2023 / 235501, and WO 2023 / 076898, each of which are incorporated by reference for all purposes herein.

[0160] 3. Guide RNA (gRNA) molecules

[0161] The terms “guide molecule,’' “guide RNA” and “gRNA” refer to any nucleic acid that promotes the specific association (or “targeting”) of an RNA-guided nuclease such as a Cas9 or a Cas12a to a target sequence such as a genomic or episomal sequence in a cell. gRNAs can be unimolecular (comprising a single RNA molecule, and referred to alternatively as chimeric), or modular (comprising more than one, and typically two, separate RNA molecules, such as a crRNA and a tracrRNA, which are usually associated with one another, for instance by duplexing). gRNAs and their component parts are described throughout the literature, for instance in Briner et al. (Molecular Cell 56(2), 333-339, October 23, 2014 (Briner), which is incorporated by reference), and in Cotta-Ramusino. The guide molecule can be an RNA molecule. The guide molecule can also comprise one or more nucleotides other than RNA nucleotides, for example, the guide molecule can be a DNA / RNA hybrid molecule, and / or the guide molecule can comprise one or more modified nucleotides (including, but not limited to, one or more modified DNA or RNA nucleotides).

[0162] In bacteria and archaea, type II CRISPR systems generally comprise an RNA-guided nuclease protein such as Cas9, a CRISPR RNA (crRNA) that comprises a 5’ region that is complementary to a foreign sequence, and a trans-activating crRNA (tracrRNA) that comprises a 5’ region that is complementary to, and forms a duplex with, a 3’ region of the crRNA. This duplex can facilitate the formation of — and is necessary for the activity of — the Cas / gRNA complex. As type II CRISPR systems were adapted for use in gene editing, it was discovered that the crRNA and tracrRNA could be joined into a single unimolecular or chimeric guide RNA, in one non-limiting example, by means of a four nucleotide (e.g., GAAA) “tetraloop” or “linker” sequence bridging complementary regions of the crRNA (at its 3’ end) and the tracrRNA (at its 5’ end). (Mali et al. Science. 2013 Feb 15; 339(6121): 823-826 (“Mali”); Jiang et al. Nat Biotechnol. 2013 Mar; 31(3): 233-239 (“Jiang”); and Jinek et al., 2012 Science Aug. 17; 337(6096): 816-821 (“Jinek”), all of which are incorporated by reference herein.) Guide RNAs. whether unimolecular or modular, comprise a “targeting domain"’ that is fully or partially complementary to a target domain within a target sequence, such as a DNA sequence in the genome of a cell where editing is desired. Targeting domains are referred to by various names in the literature, including without limitation “guide sequences” (Hsu et al., Nat Biotechnol. 2013 Sep; 31(9): 827-832, (“Hsu”), incorporated by reference herein), “complementarity regions"’ (Cotta-Ramusino). “spacers"’ (Briner) and generically as “crRNAs” (Jiang). Irrespective of the names they are given, targeting domains are typically 10-30 nucleotides in length, and in certain embodiments are 16-24 nucleotides in length (for instance, 1 , 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides in length), and are at or near the 5’ terminus of in the case of a Cas9 gRNA, and at or near the 3’ terminus in the case of a Casl2a gRNA.

[0163] In addition to the targeting domains, gRNAs typically (but not necessarily, as discussed below) comprise a plurality of domains that can influence the formation or activity of gRNA / Cas9 complexes. For instance, as mentioned above, the duplexed structure formed by first and secondary complementarity domains of a gRNA (also referred to as a repeat: antirepeat duplex) interacts with the recognition (REC) lobe of Cas9 and can mediate the formation of Cas9 / gRNA complexes. (Nishimasu et al.. Cell 156. 935-949. February 27, 2014 (Nishimasu 2014) and Nishimasu et al., Cell 162, 1113-1126, August 27, 2015 (Nishimasu 2015), both incorporated by reference herein). It should be noted that the first and / or second complementarity domains can contain one or more poly -A tracts, which can be recognized by RNA polymerases as a termination signal. The sequence of the first and second complementarity domains are, therefore, optionally modified to eliminate these tracts and promote the complete in vitro transcription of gRNAs, for instance through the use of A-G swaps as described in Briner, or A-U swaps. These and other similar modifications to the first and second complementarity domains are within the scope of the present disclosure.

[0164] Along with the first and second complementarity domains, Cas9 gRNAs typically comprise two or more additional duplexed regions that are involved in nuclease activity in vivo but not necessarily in vitro. (Nishimasu 2015). A first stem-loop near the 3’ portion of the second complementarity domain is referred to variously as the “proximal domain,” (Cotta- Ramusino) “stem loop 1” (Nishimasu 2014 and 2015) and the “nexus” (Briner). One or more additional stem loop structures are generally present near the 3’ end of the gRNA, with the number varying by species: S. pyogenes gRNAs typically comprise two 3’ stem loops (for a total of four stem loop structures including the repeat: anti-repeat duplex), while A aureus and other species have only one (for a total of three stem loop structures). A description of conserved stem loop structures (and gRNA structures more generally) organized by species is provided in Briner.

[0165] While the foregoing description has focused on gRNAs for use with Cas9, it should be appreciated that other RNA-guided nucleases have been (or can in the future be) discovered or invented which utilize gRNAs that differ in some ways from those described to this point. For instance, Casl2a (also known as Cpfl); “CRISPR from Prevotella and Franciscella 1”) is an RNA-guided nuclease that does not require a tracrRNA to function. (Zetsche et al.. 2015, Cell 163, 759-771 October 22, 2015 (Zetsche T), incorporated by reference herein). A gRNA for use in a Cas12a genome editing system generally comprises a targeting domain and a complementarity domain (alternately referred to as a "handle"). It should also be noted that, in gRNAs for use with Cas 12a, the targeting domain is usually present at or near the 3 ’ end, rather than the 5’ end as described above in connection w ith Cas9 gRNAs (the handle is at or near the 5’ end of a Cas 12a gRNA).

[0166] Those of skill in the art will appreciate that, although structural differences can exist between gRNAs from different prokaryotic species, or between Cas 12a and Cas9 gRNAs, the principles by which gRNAs operate are generally consistent. Because of this consistency of operation, gRNAs can be defined, in broad terms, by their targeting domain sequences, and skilled artisans will appreciate that a given targeting domain sequence can be incorporated in any suitable gRNA, including a unimolecular or chimeric gRNA, or a gRNA that comprises one or more chemical modifications and / or sequential modifications (substitutions, additional nucleotides, truncations, etc.). Thus, for economy of presentation in this disclosure, gRNAs can be described solely in terms of their targeting domain sequences.

[0167] More generally, skilled artisans will appreciate that some aspects of the present disclosure relate to systems, methods and compositions that can be implemented using multiple RNA-guided nucleases. For this reason, unless otherwise specified, the term gRNA should be understood to encompass any suitable gRNA that can be used with any RNA-guided nuclease, and not only those gRNAs that are compatible w ith a particular RNA-guided nuclease, e.g., a particular species of Cas9 or Cas 12a. By way of illustration, the term gRNA can, in certain embodiments, comprise a gRNA for use with any RNA-guided nuclease occurring in a Class 2 CRISPR system, such as a type II or type V CRISPR system, or an RNA-guided nuclease derived or adapted therefrom.

[0168] In some embodiments, the gRNA scaffold sequence (e.g., SEQ ID NO: 69; Tables 7A- 7C) is the same sequence as the gRNA hairpin region (e.g., SEQ ID NO: 252; Table 26). In some embodiments, the guide RNA used comprises a modification as compared to the standard gRNA scaffold. Such modifications may comprise, for example, chemical modifications of a part of the gRNA, e.g., of a nucleobase or backbone moiety. In some embodiments, such a modification may also comprise the presence of one or more DNA nucleotide within the gRNA, e.g., within or outside of the targeting domain. In some embodiments, the modification may comprise an extension of the gRNA scaffold, e.g., by addition of 1-100 nucleotides, including RNA and / or DNA nucleotides at the 3’ or the 5’ terminus of the guide RNA, e.g., at the terminus distal to the targeting domain.

[0169] In certain embodiments, a gRNA complexed to an unmodified or modified Casl2a protein may be modified to increase the editing efficiency of a target nucleic acid. In certain embodiments, the modified gRNA may comprise one or more modifications including a phosphorothioate (PS2) linkage modification, a 2’-O-methyl modification (non-limiting exemplary modifications are illustrated in FIG. 3), one or more or a stretch of additional nucleotides (e.g., RNA or deoxyribonucleic acid (DNA) nucleotides) not found in a corresponding native gRNA (also referred herein as a “gRNA extension’'), or combinations thereof.

[0170] In some embodiments, a gRNA used herein comprises one or more or a stretch of additional ribonucleic acid or deoxyribonucleic acid (DNA) bases outside of the spacer region, also referred to herein as a “gRNA extension.” In some embodiments, a gRNA used herein comprises a gRNA extension that comprises one or more or a stretch of DNA bases, referred to herein as a “DNA extension”. In some embodiments, a gRNA used herein comprises a DNA extension at the 5' end of the gRNA, the 3' end of the gRNA, or a combination thereof. In certain embodiments, the DNA extension may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,

[0171] 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 32, 33, 34, 35, 36, 37, 38, 39, 40,

[0172] 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57. 58. 59. 60, 61, 62, 63, 64, 65,

[0173] 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,

[0174] 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 DNA bases long. For example, in certain embodiments, the DNA extension may be 1, 2, 3, 4, 5, 10, 15, 20, or 25 DNA bases long. In certain embodiments, the DNA extension may comprise one or more DNA bases selected from adenine (A), guanine (G). cytosine (C), or thymine (T). In certain embodiments, the DNA extension comprises the same DNA bases. For example, the DNA extension may comprise a stretch of adenine (A) bases. In certain embodiments, the DNA extension may comprise a stretch of thymine (T) bases. In certain embodiments, the DNA extension comprises a combination of different DNA bases. In certain embodiments, a DNA extension may comprise or consist of a sequence set forth in Table 3. In certain embodiments, a gRNA used herein comprises a DNA extension as well as one or more phosphorothioate linkage modifications, one or more phosphorodi thioate (PS2) linkage modifications, one or more 2’-O-methyl modifications, or combinations thereof. In certain embodiments, the one or more modifications may be at the 5’ end of the gRNA, at the 3’ end of the gRNA, or combinations thereof. In certain embodiments, a gRNA including a DNA extension may comprise a sequence set forth in Table 3 that comprises a DNA extension. Without wishing to be bound by theory, it is contemplated that any DNA extension may be used herein, so long as it does not hybridize to the target nucleic acid being targeted by the gRNA. In some embodiments the DNA extension additionally exhibits an increase in editing efficiency, e.g., via changes to gRNA stability , uptake, and / or activity', at the target nucleic acid site relative to a gRNA which does not comprise such a DNA extension.

[0175] In some embodiments, a gRNA used herein comprises a gRNA extension that comprises one or more or a stretch of ribonucleic acid (RNA) bases, also referred to herein as an “RNA extension.” In some embodiments, a gRNA used herein comprises an RNA extension at the 5‘ end of the gRNA. the 3’ end of the gRNA. or a combination thereof. In certain embodiments, the RNA extension may be 1. 2, 3, 4. 5. 6, 7, 8. 9, 10, 11. 12. 13. 14. 15. 16. 17,

[0176] 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,

[0177] 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,

[0178] 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84. 85, 86, 87, 88, 89, 90, 91, 92,

[0179] 93, 94, 95, 96, 97, 98, 99, or 100 RNA bases long. For example, in certain embodiments, the RNA extension may be 1, 2, 3, 4, 5, 10, 15, 20, or 25 RNA bases long. In certain embodiments, the RNA extension may comprise one or more RNA bases selected from adenine (rA), guanine (rG), cytosine (rC), or uracil (rU), in which the “r” represents RNA, 2'-hydroxy. In certain embodiments, the RNA extension comprises the same RNA bases. For example, the RNA extension may comprise a stretch of adenine (rA) bases. In certain embodiments, the RNA extension comprises a combination of different RNA bases. In certain embodiments, an RNA extension may comprise or consist of a sequence set forth in Table 3. In certain embodiments, a gRNA used herein comprises an RNA extension as well as one or more phosphorothioate linkage modifications, one or more phosphorodithioate (PS2) linkage modifications, one or more 2’ -O-methyl modifications, or combinations thereof. In certain embodiments, the one or more modifications may be at the 5’ end of the gRNA, at the 3’ end of the gRNA, or combinations thereof. In certain embodiments, a gRNA including an RNA extension may comprise a sequence set forth in Table 3 that comprises an RNA extension. gRNAs including an RNA extension at the 5’ end of the gRNA may comprise a sequence disclosed herein. gRNAs including an RNA extension at the 3’ end of the gRNA may comprise a sequence disclosed herein.

[0180] It is contemplated that gRNAs used herein may also comprise an RNA extension and a DNA extension. In certain embodiments, the RNA extension and DNA extension may both be at the 5’ end of the gRNA, the 3’ end of the gRNA, or a combination thereof. In certain embodiments, the RNA extension is at the 5‘ end of the gRNA and the DNA extension is at the 3’ end of the gRNA. In certain embodiments, the RNA extension is at the 3’ end of the gRNA and the DNA extension is at the 5’ end of the gRNA.

[0181] It is further contemplated that gRNAs used herein may comprise a gRNA extension that is a hybrid extension that comprises both deoxyribonucleic acid and ribonucleic acid moieties.

[0182] In some embodiments, a gRNA which comprises a modification, e.g., a DNA extension at the 5’ end, is complexed with a RNA-guided nuclease, e.g., an AsCasl2a nuclease, to form an RNP complex, (such RNP complex formation occurring either prior to delivery of a composition described herein to a subject or following such delivery, e.g., in a cell after expression of an mRNA encoding the RNA-guided nuclease), which then edits a target cell (e.g., a liver cell). Exemplary suitable 5’ extensions for guide RNAs, e.g., Cas 12a guide RNAs, are provided in the table below:

[0183] Table 3: gRNA 5’ Extensions

[0184] All bases are in upper case Lowercase “r” represents RNA, 2’-hydroxy; bases not modified by an “r” are DNA

[0185] All bases are linked via standard phosphodiester bonds except as noted:

[0186] “*” represents phosphorothioate modification

[0187] "PS " represents phosphorothioate modification

[0188] Additional suitable gRNA modifications will be apparent to those of ordinary skill in the art based on the present disclosure. Suitable gRNA modifications comprise, for example, those described in PCT application PCT / US2018 / 054027, filed on Oct. 2. 2018, and entitled “MODIFIED CPF1 GUIDE RNA;” in PCT application PCT / US2015 / 000143, filed on Dec. 3, 2015, and entitled “GUIDE RNA WITH CHEMICAL MODIFICATIONS;” in PCT application PCT / US2016 / 026028, filed Apr. 5. 2016, and entitled “CHEMICALLY MODIFIED GUIDE RNAS FOR CRISPR / CAS-MEDIATED GENE REGULATION;” and in PCT application PCT / US2016 / 053344, filed on Sep.23, 2016, and entitled “NUCLEASE- MEDIATED GENOME EDITING OF PRIMARY CELLS AND ENRICHMENT THEREOF;” the entire contents of each of which are incorporated herein by reference. Without being bound by theory, in certain embodiments of genome editing systems of the present disclosure, the one or more modifications of the gRNA enhance binding affinity of the gRNA molecule to RNA-guided nuclease of the genome editing system, e.g., a Casl2a nuclease.

[0189] 4. Guide RNA design

[0190] Methods for selection and validation of target sequences as well as off-target analyses have been described previously, e.g., in Mali; Hsu; Fu et al., 2014 Nat Biotechnol 32(3): 279- 84. Heigwer et al.. 2014 Nat methods 11(2): 122-3; Bae et al. (2014) Bioinformatics 30(10): 1473-5; and Xiao A et al. (2014) Bioinformatics 30(8): 1180-1 182. Each of these references is incorporated by reference herein. In certain non-limiting embodiments, gRNA design can involve the use of a software tool to optimize the choice of potential target sequences corresponding to a user’s target sequence, e.g., to minimize total off-target activity across the genome. These and other guide selection methods are described in detail in Maeder and Cotta- Ramusino.

[0191] In certain embodiments, one or more or all of the nucleotides in a gRNA are modified. Strategies for modifying a gRNA are described in WO2019 / 152519, published Aug. 8, 2019, the entire contents of which are expressly incorporated herein by reference.

[0192] Non-limiting examples of guide RNAs suitable for certain embodiments embraced by the present disclosure are provided herein, for example, in the Tables below. Those of ordinary skill in the art will be able to envision suitable guide RNA sequences for a specific nuclease, e.g., a Cas9 or Casl2a nuclease, from the disclosure of the targeting domain sequence, either as a DNA or RNA sequence. For example, a guide RNA comprising a targeting sequence consisting of RNA nucleotides would comprise the RNA sequence corresponding to the targeting domain sequence provided as a DNA sequence, and thus contain uracil instead of thymidine nucleotides. For example, a guide RNA comprising a targeting domain sequence consisting of RNA nucleotides and described by the DNA sequence TGGGGTCCGACTATGCTGGTG (SEQ ID NO: 24) would have a targeting domain of the corresponding RNA sequence rUrGrGrGrGrUrCrCrGrArCrUrArUrGrCrUrGrGrUrG (SEQ ID NO: 39). As will be apparent to the skilled artisan, such a targeting sequence would be linked to a suitable guide RNA scaffold, e.g., a crRNA scaffold sequence or a chimeric crRNA / tracrRNA scaffold sequence. Suitable gRNA scaffold sequences are known to those of ordinary skill in the art. For AsCasl2a, for example, a suitable scaffold sequence comprises the sequence rUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArU (SEQ ID NO: 69), added to the 5 ’-terminus of the targeting domain. In the example above, this would result in a Casl2a guide RNA of the sequence; rUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrGrGrGrGrUrCrCrGrArCrUrA rUrGrCrUrGrGrUrG (SEQ ID NO: 54). Those of skill in the art would further understand how to modify such a guide RNA. For example, adding a 25-mer DNA extension (e.g., SEQ ID NO: 7) would result, for example, in a guide RNA of the sequence ATGTGTTTTTGTCAAAAGACCTTTT rUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrGrGrGrGrUrCrCrGrArCrUrArUrGrC rUrGrGrUrG (SEQ ID NO: 70). It will be understood that the exemplary targeting sequences provided herein are not limiting, and additional suitable sequences, e.g., variants of the specific sequences disclosed herein, will be apparent to the skilled artisan based on the present disclosure in view of the general knowledge in the art.

[0193] In some embodiments, the gRNA for use in the disclosure is a gRNA targeting a liver- expressed gene. In certain embodiments, the liver-expressed gene is involved in regulating lipid and / or cholesterol metabolism. In certain embodiments, decreasing the expression of the liver-expressed gene decreases the levels of circulating LDL. In certain embodiments, decreasing the expression of the liver-expressed gene decreases the levels of circulating Lp(a). In certain embodiments, the liver-expressed gene is LPA, ANGPTL3, PCSK9. LDLR. APOC2, APOC3, APOB. MTP, ANGPTL4, ANGPTL8, APOA5, ApoB, APOE, IDOL, NPC1L1, ASGR1, TM6SF2, GALNT2, LPL, MLXIPL, SORT1, TRIBI, MARC1, ABCG5, ABCG8, PNPLA3, TM6SF2, HFE. HMOX-1, UGT1A1, STAP1, LDLRAP1, LMF-1, GP1HBP1, CYP27A1, LIPA or ATP7B.

[0194] In some embodiments, the gRNA for use in the disclosure is a gRNA targeting LPA (LPA gRNA). In some embodiments, the target sequence of an LPA gene comprises or consists of a nucleotide sequence set forth in SEQ iD NOs: 24-38 and 91 (Table 4A). In certain embodiments, the target sequence of an LPA gene comprises or consists of the nucleotide sequence set forth in SEQ iD NOs: 24 or 26. In some embodiments, the targeting domain of the gRNA molecule comprises or consists of a nucleotide sequence set forth in SEQ iD NOs: 39-53 and 92 (Table 5A). In certain embodiments, the targeting domain of the gRNA targeting an LPA gene is SEQ ID NO: 39, or SEQ ID NO: 41. In some embodiments, the gRNA molecule targeting an LPA gene comprises or consists of a nucleotide sequence set forth in SEQ iD NOs: 54-68 (Table 6A). In certain embodiments, the gRNA molecule targeting an LPA gene comprises or consists of the sequence set forth in SEQ ID NO: 54, or SEQ ID NO: 56. An exemplary LPA gene target sequence, gRNA targeting domain, scaffold sequence, and DNA extension are set forth in Table 7. In some embodiments, the gRNA for use in the disclosure is a gRNA targeting ANGPTL3 (ANGPTL3 gRNA). In some embodiments, the target sequence of an ANGPTL3 gene comprises or consists of a nucleotide sequence set forth in SEQ iD NOs: 310-320 (Table 4B). In certain embodiments, the target sequence of an LPA gene comprises or consists of the nucleotide sequence set forth in SEQ iD NOs: 312 OR 317. In some embodiments, the targeting domain of the gRNA molecule comprises or consists of a nucleotide sequence set forth in SEQ iD NOs: 336-346 (Table 5B). In certain embodiments, the targeting domain of the gRNA targeting an LPA gene is SEQ ID NO: 338 or SEQ ID NO: 343. In some embodiments, the gRNA molecule targeting m ANGPTI.3 gene comprises or consists of a nucleotide sequence set forth in SEQ iD NOs: 362-372 (Table 6B). In certain embodiments, the gRNA molecule targeting an ANGPTL3 gene comprises or consists of the sequence set forth in SEQ ID NO: 364, or SEQ ID NO: 369. An exemplary ANGPTL3 gene target sequence, gRNA targeting domain, scaffold sequence, and DNA extension are set forth in Table 7B.

[0195] In some embodiments, the gRNA for use in the disclosure is a gRNA targeting PCSK9 (PCSK9 gRNA). Human PCSK9 gene is located on Chromosome 1. The PCSK9 transcript ENST00000302118.5 comprises twelve exons that encode the PCSK9 protein. Expression of PCSK9 degrades the receptor for low-density lipoprotein particles (LDLR), thereby increasing V / LDL levels, and thereby increasing Lp(a) levels. In contrast, PCSK9 deficiencies are associated with reduction of all plasma lipoproteins. Heterozy gous carriers of PCSK9 loss of function variants have reduced plasma levels of LDL and are at lower risk of developing cardiovascular disease, as compared to non-carriers. In view of such outcomes, treatment with an PSCK9-blocking antibody (alirocumab and evolocumab) has been evaluated and found to reduce LDL-C. In some embodiments, the target sequence of a PCSK9 gene comprises or consists of a nucleotide sequence set forth in SEQ iD NOs: 321-335 (Table 4C). In certain embodiments, the target sequence of a PCSK9 gene compnses or consists of the nucleotide sequence set forth in SEQ ID NOs: 334-335. In some embodiments, the targeting domain of the gRNA molecule comprises or consists of a nucleotide sequence set forth in SEQ iD NOs: 347-361 (Table 5C). In certain embodiments, the targeting domain of the gRNA targeting a PCSK9 gene is SEQ ID NO: 360, or SEQ ID NO: 361. In some embodiments, the gRNA molecule targeting a PCSK9 gene comprises or consists of a nucleotide sequence set forth in SEQ iD NOs: 373-387 (Table 6C). In certain embodiments, the gRNA molecule targeting a PCSK9 gene comprises or consists of the sequence set forth in SEQ ID NO: 386, or SEQ ID NO: 387. An exemplary PCSK9 gene target sequence. gRNA targeting domain, scaffold sequence, and DNA extension are set forth in Table 7C. Table 4A: LPA Target Sequences

[0196] 'Chromosome 6, LPA transcript = ENST00000316300.10

[0197] Table 4B: ANGPTL3 Target Sequences

[0198] 'Chromosome 1,ANGPTL3 transcript = ENST00000371129.4

[0199] Table 4C: PCSK9 Target Sequences

[0200] 'Chromosome 1, PCSK9 transcript = ENST00000302118.5 Table 5A: LPA Targeting Sequences

[0201] Table 5B: ANGPTL3 Targeting Sequences

[0202] Table 5C: PCSK9 Targeting Sequences

[0203] Table 6B; ANGPTL3 gRNA sequences

[0204] Table 6C: PCSK9 gRNA sequences

[0205] Table 7A: Exemplary LPA guide RNA (DNA / RNA oligonucleotide) All bases are in upper case

[0206] Lowercase “r” represents RNA, 2’-hydroxy; bases not modified by an "‘r” are DNA All linkages between the nucleotides are standard phosphodiester groups.

[0207] All bases are in upper case

[0208] Lowercase “r” represents RNA, 2’-hydroxy; bases not modified by an “r” are DNA All linkages between the nucleotides are standard phosphodiester groups.

[0209] All bases are in upper case

[0210] Lowercase "‘r” represents RNA, 2’-hydroxy; bases not modified by an “r” are DNA All linkages between the nucleotides are standard phosphodiester groups. In some embodiments, the targeting domain of the gRNA molecule has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to a nucleotide sequence set forth in SEQ iD NOs: 39-53, 336-346, or 347-361. In some embodiments, the targeting domain of the gRNA molecule has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations relative to a nucleotide sequence set forth in SEQ iD NOs: 39-53, 336-346, or 347-361. In some embodiments, the targeting domain of the gRNA molecule has less than 2, 3, 4. 5, 6, 7. 8, 9, or 10 mutations relative to a nucleotide sequence set forth in SEQ iD NOs: 39-53, 336-346, or 347-361.

[0211] 4. 1 Guide RNA modifications

[0212] The activity, stability, or other characteristics of gRNAs can be altered through the incorporation of certain modifications. As one example, transiently expressed or delivered nucleic acids can be prone to degradation by, e.g, cellular nucleases. Accordingly, the gRNAs described herein can contain one or more modified nucleosides or nucleotides which introduce stability toward nucleases. While not wishing to be bound by theory it is also believed that certain modified gRNAs described herein can exhibit a reduced innate immune response when introduced into cells. Those of skill in the art will be aware of certain cellular responses commonly observed in cells, e.g.. mammalian cells, in response to exogenous nucleic acids, particularly those of viral or bacterial origin. Such responses, which can comprise induction of cytokine expression and release and cell death, can be reduced or eliminated altogether by the modifications presented herein.

[0213] Certain exemplary modifications discussed in this section can be included at any position within a gRNA sequence including, without limitation at or near the 5’ end (e.g , within 1-10, 1-5, or 1-2 nucleotides of the 5’ end) and / or at or near the 3’ end (e.g, within 1- 10, 1-5, or 1-2 nucleotides of the 3’ end). In some cases, modifications are positioned within functional motifs, such as the repeat-anti-repeat duplex of a Cas9 gRNA, a stem loop structure of a Cas9 or Casl2a gRNA, and / or a targeting domain of a gRNA.

[0214] As one example, the 5’ end of a gRNA can comprise a eukaryotic mRNA cap structure or cap analog (e.g, G(5 )ppp(5)G cap analog, am 7G(5 )ppp(5 )G cap analog, or a 3 ’-O-Me- m7G(5 )ppp(5 )G anti reverse cap analog (ARCA)), as shown below:

[0215]

[0216] The cap or cap analog can be included during either chemical synthesis or in vitro transcription of the gRNA.

[0217] Along similar lines, the 5’ end of the gRNA can lack a 5’ triphosphate group. For instance, in vitro transcribed gRNAs can be phosphatase-treated (e.g, using calf intestinal alkaline phosphatase) to remove a 5‘ triphosphate group.

[0218] Another modification involves the addition, at the 3’ end of a gRNA. of a plurality (e.g. , 1-10, 10-20, or 25-200) of adenine (A) residues referred to as a poly A tract. The poly A tract can be added to a gRNA during chemical synthesis, following in vitro transcription using a polyadenosine polymerase (e.g., E. coli Poly(A)Polymerase), or in vivo by means of a poly adenylation sequence, as described in Maeder.

[0219] It should be noted that the modifications described herein can be combined in any suitable manner, e.g., a gRNA, wh ether transcribed in vivo from a DNA vector, or in vitro transcribed gRNA, can comprise either or both of a 5‘ cap structure or cap analog and a 3’ polyA tract.

[0220] Guide RNAs can be modified at a 3’ terminal U ribose. For example, the two terminal hydroxyl groups of the U ribose can be oxidized to aldehyde groups and a concomitant opening of the ribose ring to afford a modified nucleoside as shown below: wherein “U” can be an unmodified or modified uridine.

[0221] The 3’ terminal U ribose can be modified with a 2’3’ cyclic phosphate as shown below: wherein “U” can be an unmodified or modified uridine.

[0222] Guide RNAs can contain 3’ nucleotides which can be stabilized against degradation, e.g., by incorporating one or more of the modified nucleotides described herein. In certain embodiments, uridines can be replaced with modified uridines, e.g., 5-(2-amino)propyl uridine, and 5 -bromo uridine, or with any of the modified uridines described herein; adenosines and guanosines can be replaced with modified adenosines and guanosines, e.g., with modifications at the 8-position, e.g, 8-bromo guanosine, or with any of the modified adenosines or guanosines described herein.

[0223] In certain embodiments, sugar-modified ribonucleotides can be incorporated into the gRNA, e.g., wherein the 2’ OH-group is replaced by a group selected from H, -OR, -R (wherein R can be. e.g, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), halo, -SH, -SR (wherein R can be, e.g, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), amino (wherein amino can be, e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroaryl amino, or amino acid); or cyano (-CN). In certain embodiments, the phosphate backbone can be modified as described herein, e.g, with a phosphothioate (PhTx) group. In certain embodiments, one or more of the nucleotides of the gRNA can each independently be a modified or unmodified nucleotide including, but not limited to 2’-sugar modified, such as, 2’-O-methyl, 2’-O-methoxyethyl, or 2’-Fluoro modified including, e.g., 2’-F (as illustrated in FIG. 3) or 2’-O-methyl, adenosine (A), 2’-F or 2’-O-methyl, cytidine (C), 2’-F or 2'-O-methyl, uridine (U), 2’-F or 2’-O-methyl, thymidine (T), 2’-F or 2’-O-methyl, guanosine (G), 2’-O- methoxyethyl-5-methyluridine (Teo), 2’ -O-methoxy ethyladenosine (Aeo), 2’-O- methoxyethyl-5-methylcytidine (m5Ceo), and any combinations thereof.

[0224] Guide RNAs can also comprise “locked’' nucleic acids (LNA) in which the 2’ OH- group can be connected, e.g., by aCl-6 alkylene or Cl -6 heteroalkylene bridge, to the 4' carbon of the same ribose sugar. Any suitable moiety can be used to provide such bridges, which may comprise without limitation methylene, propylene, ether, or amino bridges; O-amino (wherein amino can be, e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino) and aminoalkoxy or O(CH2)n-amino (wherein amino can be, e.g.. NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino).

[0225] In certain embodiments, a gRNA can comprise a modified nucleotide which is multicyclic (e.g. tricyclo; and “unlocked” forms, such as glycol nucleic acid (GNA) (e.g. R- GNA or S-GNA, where ribose is replaced by glycol units attached to phosphodiester bonds), or threose nucleic acid (TNA, where ribose is replaced with o.-L-threofuranosyl-(3’^2')).

[0226] Generally, gRNAs comprise the sugar group ribose, which is a 5-membered ring with an oxygen atom. Exemplary modified gRNAs can comprise, without limitation, replacement of the oxygen in ribose (e.g. , with sulfur (S), selenium (Se), or alkylene, such as, e.g. , methylene or ethylene); addition of a double bond (e.g., to replace ribose with cyclopentenyl or cyclohexenyl); ring contraction of ribose (e.g., to form a 4-membered ring of cyclobutane or oxetane); ring expansion of ribose (e.g. , to form a 6- or 7-membered ring having an additional carbon or heteroatom, such as for example, anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino that also has a phosphoramidate backbone). Although the majority of sugar analog alterations are localized to the 2’ position, other sites are amenable to modification, including the 4’ position. In certain embodiments, a gRNA comprises a 4’-S, 4’- Se or a 4’-C-aminomethyl-2’-O-Me modification.

[0227] In certain embodiments, deaza nucleotides, e.g., 7-deaza- adenosine, can be incorporated into the gRNA. In certain embodiments, O- and N-alkylated nucleotides, e.g., N6- methyl adenosine, can be incorporated into the gRNA. In certain embodiments, one or more or all of the nucleotides in a gRNA are deoxynucleotides.

[0228] In certain embodiments, a gRNA comprises one or more 2’F modifications. In certain embodiments, a 2’F modification is positioned on a 5‘ and / or 3‘ terminal nucleotide. In certain embodiments, a 2’F modification is position on a nucleotide internal to the gRNA (which may be represented herein asc'i2F”). In certain embodiments, the gRNA comprises a 2’F modification on one or more U nucleotide. In certain embodiments, the gRNA comprises a 2’F modification on one or more G nucleotide. In certain embodiments, the gRNA comprises a 2'F modification on one or more A nucleotide. In certain embodiments, the 2’F modification is absent in at least one U nucleotide. In certain embodiments, the 2’F modification is absent in at least one G nucleotide. In certain embodiments, the 2’F modification is absent in at least one A nucleotide.

[0229] Table 7D illustrates exemplary' combinations of 2’F modifications of the nucleotides in the exemplary hairpin sequence of SEQ ID NO: 252.

[0230] Table 7D. Potential 2'F modifications for an exemplary hairpin sequence

[0231] In certain embodiments, the modifications exemplified in the hairpin sequence are also employed in the targeting region (i.e., one or more of the terminal and / or internal nucleotides is modified as illustrated in the exemplary modifications of the hairpin sequence). For example, but not by way of limitation, the gRNA can comprise one or more 2’F modifications across the hairpin and / or targeting regions of the gRNA. In certain embodiments, the gRNA can comprise a “conservative pattern” of modifications as disclosed herein and / or an “aggressive pattern" of modifications as disclosed herein. In certain embodiments, the gRNA comprises a conservative patterned hairpin and a conservative patterned targeting region. In certain embodiments, the gRNA comprises a conservative patterned hairpin and an aggressive patterned targeting region. In certain embodiments, the gRNA comprises an aggressive patterned hairpin and a conservative patterned targeting region. In some embodiments, the gRNA comprises an aggressive patterned hairpin and an aggressive patterned targeting region. In certain embodiments, a gRNA hairpin region of the present disclosure has the same sequence as the gRNA scaffold sequence. For example, a Casl2a gRNA can have hairpin sequence of SEQ ID NO: 252 (Table 7D) and a scaffold sequence of SEQ ID NO: 69 (Table 7A). In certain embodiments, a gRNA comprising an aggressive pattern of hairpin 2’F modifications comprises a hairpin of SEQ ID NO: 427. In certain embodiments, a gRNA comprising a conservative pattern of hairpin 2’F modifications comprises a hairpin of SEQ ID NO: 421.

[0232] In certain embodiments, a gRNA as used herein may be a modified or an unmodified gRNA. In certain embodiments, a gRNA may include one or more modifications. In certain embodiments, the one or more modifications may include a phosphorothioate linkage modification, aphosphorodithioate (PS2) linkage modification, a 2’-O-methyl modification, or combinations thereof. In certain embodiments, the one or more modifications may be at the 5 ’ end of the gRNA, at the 3’ end of the gRNA. or combinations thereof. In general, any combination of modifications may be used. In non-limiting examples, patterns of modifications that can be used include: (i) a combination of 5’ extension, 5’3’ idT, and 2’F in hairpin aggressive pattern (see, e.g., the relevant gRNAs in Tables 18a and 26 for illustration of an exemplary “hairpin aggressive pattern”); (ii) a combination of 5’3' idT and 2’F in hairpin aggressive pattern; (iii) a combination of 5'3' idT and 5‘ extension; (iv) a combination of 5’ extension, 5’3’ idT, and 2’F in hairpin conservative pattern; and (v) a combination of 5’3’ idT and 2’F in hairpin conservative pattern. The pattern of modification of the gRNA, e.g., the modifications illustrated in Fig. 3 or described elsewhere herein, can be applied to guides for any targeting domain (e.g., targeting LPA. MYOC. ANGPTL3, or PCSK9) and / or for targeting genes in a tissue specific manner (e.g., a liver-cell specific manner).

[0233] In certain embodiments, the pattern of modification of the gRNA described herein can be applied to guides targeting a liver-expressed gene. In certain embodiments, the liver- expressed gene is involved in regulating lipid and / or cholesterol metabolism. In certain embodiments, decreasing the expression of the liver-expressed gene decreases the levels of circulating LDL. In certain embodiments, decreasing the expression of the liver-expressed gene decreases the levels of circulating Lp(a). In certain embodiments, the liver-expressed gene is LPA, ANGPTL3, PCSK9, LDLR. APOC2, APOC3. APOB. MTP, ANGPTL4. ANGPTL8, APOA5, ApoB. APOE, IDOL, NPC1L1, ASGR1, TM6SF2, GALNT2, LPL, MLXIPL, SORT1, TRIBI, MARC1, ABCG5, ABCG8, PNPLA3, TM6SF2, HFE, HMOX-1. UGT1A1, STAP1, LDLRAP1, LMF-1, GP1HBP1, CYP27A1, LIPA or ATP7B.

[0234] In certain embodiments, a modified gRNA as described herein comprises one or more modifications of the gRNA in the hairpin region, the targeting domain, or both. For example, but not by way of limitation, the hairpin region of such a modified gRNA can comprise SEQ ID NO: 252. In certain embodiments, the hairpin region of a modified gRNA comprises one or more 2’Fluorine modifications (e.g., see SEQ iD NOs: 421 or 427 in Table 26). In certain embodiments, the hairpin region of a modified gRNA comprises a DNA extension at the 5 ’ end of the hairpin region. In certain embodiments, the hairpin region of a modified gRNA comprises one or more 2’0-methyl modifications. For example, in certain embodiments, a modified gRNA comprises a IxPSOMe modification on a 5’ terminus and / or a 3’ terminus; in certain embodiments, a modified gRNA comprises a 3xPSOMe modification on a 5’ terminus and / or a 3’ terminus. In certain embodiments, the hairpin region of a modified gRNA comprises one or more 5’ inverted dT modifications. In certain embodiments, the hairpin region of a modified gRNA comprises one or more 3’ inverted dT modifications. In certain embodiments, the hairpin region of a modified gRNA comprises a 3’ or 5’ pseudoknot. In certain embodiments, the hairpin region of a modified gRNA comprises a 3’ pseudoknot. In certain embodiments, the hairpin region of a modified gRNA comprises a locked nucleic acid (LNA). In certain embodiments, the hairpin region of a modified gRNA comprises a LNA with a 5’ extension.

[0235] In certain embodiments, the hairpin region of a modified gRNA can comprise one or more of 5" extensions, 2'Fluonne modifications. 2’0-methyl modifications, 5’ inverted dT modifications, 3’ inverted dT modifications, a pseudoknot, or an LNA. For example, in certain embodiments, the hairpin region of amodified gRNA comprises a 5’ extension and a IxPSOMe modification on 5 ’ and 3 ’ ends. In certain embodiments, the hairpin region of a modified gRNA comprises a IxPSOMe modification on the 5’end and a 3’ pseudoknot. In certain embodiments, the hairpin region of the modified gRNAs comprises a 5 ’ extension and IxPSOMe modification on the 3 ’end only. In certain embodiments, the hairpin region of a modified gRNA comprises a 5’ extension and a conservative pattern of 2'F modifications. In certain embodiments, the hairpin region of the modified gRNAs comprises a 5’ extension and an aggressive pattern of 2’F modifications. In certain embodiments, a modified gRNA comprises a hairpin with a 5’ extension and inverted dT modifications at the 5’ and 3’ termini. In certain embodiments, a modified gRNA comprises a hairpin with a 5’ extension, inverted dT modifications at the 5’ and 3’ termini, and an LNA. In certain embodiments, a modified gRNA comprises a hairpin with a 5’ extension and an aggressive pattern of 2’ modifications, and IxPSOMe modifications on 5’ and 3’ termini. In certain embodiments, a modified gRNA comprises a hairpin with a 5’ extension and an aggressive pattern of 2’F modifications, and inverted dT modifications at the 5’ and 3' termini. In certain embodiments, the hairpin region of a modified gRNA comprises a 5’ extension and a 2’0Me modification.

[0236] In certain embodiments, the targeting domain of the gRNA can comprise one or more of 5’ extensions, 2’Fluorine modifications, 2’0-methyl modifications, 5’ inverted dT modifications, or 3’ inverted dT modifications, a pseudoknot, or an LNA. In certain embodiments the targeting domain of a gRNA comprises 2’F modifications at nucleotide positions 1, 8, 9, 10, 11, 12, 17, 19 and optionally 20 and 21. In certain embodiments the targeting domain of a gRNA comprises 2’F modifications at nucleotide positions 1, 2, 3, 7, 8, 9, 10, 11, 12, 14, 15, 17, 19 and optionally 20 and 21. In certain embodiments, the targeting domain of a gRNA comprises one or more of 2’Fluorine modifications, 2’0-methyl modifications, 5’ inverted dT modifications, or 3’ inverted dT modifications.

[0237] In any of the above embodiments, a gRNA can comprise any pattern of 2’F modifications in the hairpin (e.g., a conservative pattern or an aggressive pattern) and any pattern of 2’F modifications in the targeting domain (e g., 2'F modifications at nucleotide positions 1, 8, 9. 10. 11. 12. 17. 19 of the targeting domain; 2’F modifications at nucleotide positions 1, 2, 3, 7, 8, 9, 10, 11, 12, 14, 15, 17, 19 of the targeting domain; or no 2’F modifications in the targeting domain).

[0238] In certain embodiments, a genome editing system described herein comprises a gRNA comprising an RNA portion comprising a 2'F modification. In certain embodiments, the RNA portion comprises a 5‘ hairpin and a 3' targeting domain. In certain embodiments, a gRNA further comprises an extension region at the 5' end of the hairpin (e.g.. a DNA extension). In certain embodiments, a gRNA comprises a hairpin comprising a 2’F modification at one or more of nucleotide positions 1, 5, 6, 7, 8, 9, 10, 12, 13, 14, 16, 17, 18 or 19 of the hairpin i.e., counting from the 5’ end of the hairpin, e.g., having SEQ ID NO: 252). In certain embodiments, a gRNA comprises a hairpin comprising multiple 2’F modifications. In certain embodiments, a gRNA comprises a hairpin comprising 2’F modifications at two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more of nucleotide positions 1, 5, 6, 7, 8, 9, 10, 12, 13, 14, 16, 17, 18 or 19 of the hairpin. In certain embodiments, a gRNA comprises a hairpin comprising 2’F modifications at nucleotide positions 1. 5, 6, 7, 8. 9, 10. 12. 13, 14, 16, 17, 18 and 19 of the hairpin. In certain embodiments, a gRNA comprises a hairpin with a pattern of 2’F modifications that consists of 2’F modifications at nucleotide positions 1, 5, 6, 7, 8, 9, 10, 12, 13, 14, 16, 17, 18 and 19 of the hairpin (exemplifying an aggressive pattern of 2’F modifications). In certain embodiments, a gRNA comprises a hairpin comprising 2’F modifications at nucleotide positions 5, 6, 7. 8, 9, 10. 12. 13, 14, 16, 17, 18 and 19 of the hairpin. In certain embodiments, a gRNA comprises a hairpin with a pattern of 2’F modifications that consists of 2’F modifications at nucleotide positions 5, 6, 7, 8, 9, 10, 12, 13, 14, 16, 17, 18 and 19 of the hairpin. In certain embodiments, a gRNA comprises a hairpin that does not include a 2’F modification on at least one of nucleotide positions 7 or 8 of the hairpin. In certain embodiments, a gRNA comprises a hairpin that lacks a 2’F modification at each of nucleotide positions 7 and 8 of the hairpin. In certain embodiments, a — gRNA comprises a hairpin comprising 2’F modifications at nucleotide positions 1, 5, 6, 9, 10, 12, 13, 14, 16, 17, 18 and 19 of the hairpin. In certain embodiments, a gRNA comprises a hairpin with a pattern of 2'F modifications that consists of 2'F modifications at nucleotide positions 1, 5, 6, 9, 10, 12, 13, 14, 16, 17, 18 and 19 of the hairpin (exemplifying a conservative pattern of 2’F modifications). In certain embodiments, a gRNA comprises a hairpin comprising 2'F modifications at nucleotide positions 5, 6, 9, 10, 12, 13, 14, 16, 17, 18 and 19 of the hairpin. In certain embodiments, a gRNA comprises a hairpin with a pattern of 2’F modifications that consists of 2’F modifications at nucleotide positions 5, 6, 9, 10, 12, 13, 14, 16, 17, 18 and 19 of the hairpin. In certain embodiments, a gRNA comprises a hairpin that lacks a 2’F modification at one or more or all of nucleotide positions 2, 3, 4, 11 and 15 of the hairpin. In certain embodiments, a gRNA comprises a hairpin that lacks a 2'F modification at one or more, or all, of nucleotide positions 1, 3. 4, 11 and 15 of the hairpin. In certain embodiments, a gRNA comprises a hairpin that lacks a 2’F modification at each of nucleotide positions 2, 3, 4, 11 and 15 of the hairpin. In certain embodiments, the gRNA comprising the hairpin is compatible with a Casl2a nuclease (e.g., AsCasl2a).

[0239] Without wishing to be bound by theory, it is contemplated that, in certain embodiments, chemical modification of the gRNA can improve editing potency by enhancing binding affinity of the modified gRNA for an RNA-guided enzy me (e.g., Casl2a).

[0240] 5. RNA-guided nucleases

[0241] RNA-guided nucleases according to the present disclosure comprise, but are not limited to, naturally occurring Class 2 CRISPR nucleases such as Cas9, and Casl2a, as well as other nucleases derived or obtained therefrom. In functional terms, RNA-guided nucleases are defined as those nucleases that: (a) interact with (e.g. complex with) a gRNA; and (b) together with the gRNA, associate with, and optionally cleave or modify, a target region of a DNA that comprises (i) a sequence complementary to the targeting domain of the gRNA and, optionally, (ii) an additional sequence referred to as a “protospacer adjacent motif,” or “PAM,” which is described in greater detail below. As the following examples will illustrate, RNA-guided nucleases can be defined, in broad terms, by their PAM specificity and cleavage activity, even though variations can exist between individual RNA-guided nucleases that share the same PAM specificity or cleavage activity'. Skilled artisans will appreciate that some aspects of the present disclosure relate to systems, methods and compositions that can be implemented using any suitable RNA-guided nuclease having a certain PAM specificity and / or cleavage activity'. For this reason, unless otherwise specified, the term RNA-guided nuclease should be understood as a generic term, and not limited to any particular type (e.g., Cas9 vs. Cas l2a), species (e.g., S. pyogenes vs. S. aureus) or variation (e.g., full-length vs. truncated or split; naturally occurring PAM specificity' vs. engineered PAM specificity, etc.) of RNA-guided nuclease.

[0242] The PAM sequence takes its name from its sequential relationship to the “protospacer” sequence that is complementary' to gRNA targeting domains (or “spacers”). Together with protospacer sequences, PAM sequences define target regions or sequences for specific RNA- guided nuclease / gRNA combinations.

[0243] Various RNA-guided nucleases may require different sequential relationships between PAMs and protospacers. For example, Cas9 nucleases recognize PAM sequences that are 3’ of the protospacer, while Cas12a, on the other hand, generally recognizes PAM sequences that are 5 ’ of the protospacer.

[0244] In addition to recognizing specific sequential orientations of PAMs and protospacers,

[0245] RNA-guided nucleases can also recognize specific PAM sequences. S. aureus Cas9, for instance, recognizes a PAM sequence of NNGRRT or NNGRRV, wherein the N residues are immediately 3’ of the region recognized by the gRNA targeting domain. S. pyogenes Cas9 recognizes NGG PAM sequences. And F. novicida Casl2a recognizes a TTN PAM sequence. PAM sequences have been identified for a variety of RNA-guided nucleases, and a strategy for identifying novel PAM sequences has been described by Shmakov et al., 2015, Molecular Cell 60, 385-397, November 5, 2015. It should also be noted that engineered RNA-guided nucleases can have PAM specificities that differ from the PAM specificities of reference molecules (for instance, in the case of an engineered RNA-guided nuclease, the reference molecule can be the naturally occurring variant from which the RNA-guided nuclease is derived, or the naturally occurring variant having the greatest amino acid sequence homology to the engineered RNA- guided nuclease).

[0246] In addition to their PAM specificity, RNA-guided nucleases can be characterized by their DNA cleavage activity: naturally-occurring RNA-guided nucleases typically form DSBs in target nucleic acids but engineered variants have been produced that generate only SSBs (discussed above) Ran & Hsu, et al., Cell 154(6). 1380-1389, September 12, 2013 (Ran), incorporated by reference herein), or that that do not cut at all.

[0247] 5.1 Cas9

[0248] Cry stal structures have been determined for S. pyogenes Cas9 (Jinek 2014), and for S. aureus Cas9 in complex with a unimolecular guide RNA and a target DNA (Nishimasu 2014; Anders 2014; and Nishimasu 2015).

[0249] A naturally occurring Cas9 protein comprises two lobes: a recognition (REC) lobe and anuclease (NUC) lobe; each of which comprise particular structural and / or functional domains. The REC lobe comprises an arginine-rich bridge helix (BH) domain, and at least one REC domain e.g., a RECI domain and. optionally, a REC2 domain). The REC lobe does not share structural similarity with other known proteins, indicating that it is a unique functional domain. While not wishing to be bound by any theory, mutational analyses suggest specific functional roles for the BH and REC domains: the BH domain appears to play a role in gRNA:DNA recognition, whereas the REC domain is thought to interact with the repeat: anti -repeat duplex of the gRNA and to mediate the formation of the Cas9 / gRNA complex.

[0250] The NUC lobe comprises a RuvC domain, an HNH domain, and a PAM-interacting (PI) domain. The RuvC domain shares structural similarity to retroviral integrase superfamily members and cleaves the non-complementary (i.e., bottom) strand of the target nucleic acid. It can be formed from two or more split RuvC motifs (such as RuvC I, RuvCII, and RuvCIII in S. pyogenes and S. aureus). The HNH domain, meanwhile, is structurally similar to HNN endonuclease motifs, and cleaves the complementary (z. e. , top) strand of the target nucleic acid. The PI domain, as its name suggests, contributes to PAM specificity.

[0251] While certain functions of Cas9 are linked to (but not necessarily fully determined by) the specific domains set forth above, these and other functions can be mediated or influenced by other Cas9 domains, or by multiple domains on either lobe. For instance, in S. pyogenes Cas9, as described in Nishimasu 2014, the repeat: antirepeat duplex of the gRNA falls into a groove between the REC and NUC lobes, and nucleotides in the duplex interact with amino acids in the BH, PI, and REC domains. Some nucleotides in the first stem loop structure also interact with amino acids in multiple domains (PI, BH and RECI), as do some nucleotides in the second and third stem loops (RuvC and PI domains).

[0252] 5.2 Casl2a (formerly known as Cpfl)

[0253] The crystal structure of Acidaminococcus sp. Cas12a in complex with crRNA and a double-stranded (ds) DNA target including a TTTN PAM sequence has been solved by Yamano et al. (Cell. 2016 May 5; 165(4): 949-962 (Yamano), incorporated by reference herein). Casl2a, like Cas9, has two lobes: a REC (recognition) lobe, and a NUC (nuclease) lobe. The REC lobe comprises RECI and REC2 domains, which lack similarity to any known protein structures. The NUC lobe, meanwhile, comprises three RuvC domains (RuvC -I, -II and -III) and a BH domain. However, in contrast to Cas9, the Casl2a REC lobe lacks an HNH domain, and comprises other domains that also lack similarity to known protein structures: a structurally unique PI domain, three Wedge (WED) domains (WED-I. -II and -III), and a nuclease (Nuc) domain.

[0254] While Cas9 and Casl2a share similarities in structure and function, it should be appreciated that certain C as 12a activities are mediated by structural domains that are not analogous to any Cas9 domains. For instance, cleavage of the complementary strand of the target DNA appears to be mediated by the Nuc domain, which differs sequentially and spatially from the HNH domain of Cas9. Additionally, the non-targeting portion of Casl2a gRNA (the handle) adopts a pseudoknot structure, rather than a stem loop structure formed by the repeat: antirepeat duplex in Cas9 gRNAs.

[0255] Non-limiting examples of RNA-guided nucleases include. Cas9 (e.g, SpCas9, SaCas9, (KKH) SaCas9, eSpCas9, Cas9-HF1, HypaCas9, dCas9-Fokl, Sniper-Cas9, xCas9, evoCas9, SpCas9-NG, VRQR, VRER, NmeCas9, CjCas9), Casl2a (e.g., AsCasl2a, LbCasl2a), Casl2b (e.g, AaCasl2b, BhCasl2b, BhCasl2bV4), Casl2c (e.g., Casl2cl, Casl2c2), Casl2h (e.g. , Casl2hl), Casl2i (e.g, Casl2il), CasX, CasY, and CaΦ. 5.3 Modifications of RNA-guided nucleases

[0256] The RNA-guided nucleases described above have activities and properties that can be useful in a variety of applications, but the skilled artisan will appreciate that RNA-guided nucleases can also be modified in certain instances, to alter cleavage activity, PAM specificity, or other structural or functional features.

[0257] Turning first to modifications that alter cleavage activity, mutations that reduce or eliminate the activity of domains within the NUC lobe have been described above. Exemplary mutations that can be made in the RuvC domains, in the Cas9 HNH domain, or in the Cas l2a Nuc domain are described in Ran and Yamano, as well as in Cotta-Ramusino. In general, mutations that reduce or eliminate activity in one of the two nuclease domains result in RNA- guided nucleases with nickase activity, but it should be noted that the type of nickase activity varies depending on which domain is inactivated. As one example, inactivation f a RuvC domain or of a Cas9 HNH domain results in a nickase.

[0258] Modifications of PAM specificity relative to naturally occurring Cas9 reference molecules have been described by Kleinstiver et al. for both S pyogenes (Kleinstiver et al., Nature. 2015 Jul 23;523(7561):481-5 (Kleinstiver I)) and 5. aureus (Kleinstiver et al., Nat Biotechnol. 2015 Dec; 33(12): 1293-1298 (Klienstiver II)). Kleinstiver et al. have also described modifications that improve the targeting fidelity of Cas9 (Nature, 2016 January 28; 529, 490-495 (Kleinstiver III)). Each of these references is incorporated by reference herein.

[0259] RNA-guided nucleases have been split into two or more parts, as described by Zetsche et al. (Nat Biotechnol. 2015 Feb;33(2): 139-42 (Zetsche II), incorporated by reference), and by Fine et al. (Sci Rep. 2015 Jul l;5: 10777 (Fine), incorporated by reference).

[0260] RNA-guided nucleases can be, in certain embodiments, size-optimized or truncated, for instance via one or more deletions that reduce the size of the nuclease while still retaining gRNA association, target and PAM recognition, and cleavage activities. In certain embodiments, RNA guided nucleases are bound, covalently or non-covalently, to another polypeptide, nucleotide, or other structure, optionally by means of a linker. Exemplary' bound nucleases and linkers are described by Guilinger et al., Nature Biotechnology 32, 577-582 (2014). which is incorporated by reference for all purposes herein.

[0261] RNA-guided nucleases also optionally comprise a tag, such as, but not limited to, a nuclear localization signal (NLS) to facilitate movement of RNA-guided nuclease protein into the nucleus. In certain embodiments, the RNA-guided nuclease can incorporate C- and / or N- terminal nuclear localization signals, e.g., SEQ ID NO: 71, presented herein. Nuclear localization sequences are known in the art and are described in Maeder and elsewhere. The foregoing list of modifications is intended to be exemplary in nature, and the skilled artisan will appreciate, in view of the instant disclosure, that other modifications can be possible or desirable in certain applications. For brevity, therefore, exemplary systems, methods and compositions of the present disclosure are presented with reference to particular RNA-guided nucleases, but it should be understood that the RNA-guided nucleases used can be modified in ways that do not alter their operating principles. Such modifications are within the scope of the present disclosure.

[0262] Exemplary suitable nuclease variants comprise, but are not limited to, AsCas l2a variants comprising an M537R substitution, an H800A substitution, and / or an F870L substitution, or any combination thereof (numbering scheme according to AsCasl2a wild-ty pe sequence). Other suitable modifications of the AsCasl2a amino acid sequence are known to those of ordinary skill in the art. Some non-limiting exemplary sequences of wild-type AsCasl2a and AsCasl2a variants are as follows:

[0263] Exemplary His-AsCpfl-sNLS-sNLS H800A amino acid sequence:

[0264] MGHHHHHHGSTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARN DHYKELKPI1DR1YKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATY RNAIHDYFIGRTDNLTDAfNKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALL RSFDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLR EHFENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNE VLNLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLL RNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTG KITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTL KKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNK ARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQ KGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILL SNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSK YTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQI YNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAA RLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHE IIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYIT VIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQV IHEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYP AEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIK NHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQ FDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLE NDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMD ADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQELRNGSPK KKRKVGSPKKKRKV [SEQ ID NO: 71]

[0265] Exemplary Casl2a variant 1 amino acid sequence:

[0266] MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPII DRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFI GRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTY FSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKK AIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQK NDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLET AEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEK VQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEIL KSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNYATKK PYSVEKFKLNFQRPTLASGWDVNKEKNNGA1LFVKNGLYYLG1MPKQKGRYKALSF EPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEIT KEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLS SLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGH HGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNK KLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSD KFLFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKIL EQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMI HYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVL NPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFL EGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFI AGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDT MVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHI ALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQELRNGRSSDDEATADSQHAAPPK KKRKVGGSGGSGGSGGSGGSGGSGGSGGSLEHHHHHH [SEQ ID NO: 72]

[0267] Exemplary AsCas12a variant 2 amino acid sequence:

[0268] MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPII DRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFI GRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTY FSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKK AIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQK NDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLET AEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEK VQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEIL KSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNYATKK PYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSF EPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEIT KEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLS

[0269] SLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGH HGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNK KLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSD KFFFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKIL EQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMI HYQAVVVLENLNFGFKSKRTG1AEKAVYQQFEKML1DKLNCLVLKDYPAEKVGGVL NPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFL EGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFI AGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDT MVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHI

[0270] ALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQELRNGRSSDDEATADSQHAAPPK KKRKVGGSGGSGGSGGSGGSGGSGGSGGSLEHHHHHH [SEQ ID NO: 73]

[0271] Exemplary AsCas 12a variant 3 amino acid sequence:

[0272] MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPI IDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYF IGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTT YFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVK KAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAI QKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENV LETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKS AKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQE EKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARN YATKKPYSVEKFKLNFQRPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGR YKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNN FIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYT KTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIY NKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAAR LGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHE IIKDRRFTSDKFLFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYI TVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLS QVIHETVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLK DYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVW KTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFE KNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNI LPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQN PEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQELRNGRS SDDEATADSQHAAPPKKKRKVGGSGGSGGSGGSGGSGGSGGSGGSLEHHHHHH [SEQ ID NO: 74]

[0273] Exemplary AsCas 12a variant 4 amino acid sequence:

[0274] MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPI IDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYF IGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTT YFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVK KAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAI QKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENV LETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKS AKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQE EKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARN YATKKPYSVEKFKLNFQRPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGR YKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNN FIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYT KTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIY NKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAAR LGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHE IIKDRRFTSDKFLFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYI TVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLS QVIHEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLK

[0275] DYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVW

[0276] KTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFE

[0277] KNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNI

[0278] LPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQN PEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQELRNGRS SDDEATADSQHAAPPKKKRKV [SEQ ID NO: 75]

[0279] Exemplary AsCas12a variant 4 nucleotide sequence:

[0280] ATGACCCAGTTTGAAGGTTTCACCAATCTGTATCAGGTTAGCAAAACCCT

[0281] GCGTTTTGAACTGATTCCGCAGGGTAAAACCCTGAAACATATTCAAGAACAGGG

[0282] CTTCATCGAAGAGGATAAAGCACGTAACGATCACTACAAAGAACTGAAACCGAT

[0283] TATCGACCGCATCTATAAAACCTATGCAGATCAGTGTCTGCAGCTGGTTCAGCTG

[0284] GATTGGGAAAATCTGAGCGCAGCAATTGATAGTTATCGCAAAGAAAAAACCGAA

[0285] GAAACCCGTAATGCACTGATTGAAGAACAGGCAACCTATCGTAATGCCATCCAT

[0286] GATTATTTCATTGGTCGTACCGATAATCTGACCGATGCAATTAACAAACGTCACG

[0287] CCGAAATCTATAAAGGCCTGTTTAAAGCCGAACTGTTTAATGGCAAAGTTCTGAA

[0288] ACAGCTGGGCACCGTTACCACCACCGAACATGAAAATGCACTGCTGCGTAGCTTT

[0289] GATAAATTCACCACCTATTTCAGCGGCTTTTATGAGAATCGCAAAAACGTGTTTA

[0290] GCGCAGAAGATATTAGCACCGCAATTCCGCATCGTATTGTGCAGGATAATTTCCC

[0291] GAAATTCAAAGAGAACTGCCACATTTTTACCCGTCTGATTACCGCAGTTCCGAGC

[0292] CTGCGTGAACATTTTGAAAACGTTAAAAAAGCCATCGGCATCTTTGTTAGCACCA

[0293] GCATTGAAGAAGTTTTTAGCTTCCCGTTTTACAATCAGCTGCTGACCCAGACCCA

[0294] GATTGATCTGTATAACCAACTGCTGGGTGGTATTAGCCGTGAAGCAGGCACCGA

[0295] AAAAATCAAAGGTCTGAATGAAGTGCTGAATCTGGCCATTCAGAAAAATGATGA

[0296] AACCGCACATATTATTGCAAGCCTGCCGCATCGTTTTATTCCGCTGTTCAAACAA

[0297] ATTCTGAGCGATCGTAATACCCTGAGCTTTATTCTGGAAGAATTCAAATCCGATG

[0298] AAGAGGTGATTCAGAGCTTTTGCAAATACAAAACGCTGCTGCGCAATGAAAATG

[0299] TTCTGGAAACTGCCGAAGCACTGTTTAACGAACTGAATAGCATTGATCTGACCCA

[0300] CATCTTTATCAGCCACAAAAAACTGGAAACCATTTCAAGCGCACTGTGTGATCAT

[0301] TGGGATACCCTGCGTAATGCCCTGTATGAACGTCGTATTAGCGAACTGACCGGTA

[0302] AAATTACCAAAAGCGCGAAAGAAAAAGTTCAGCGCAGTCTGAAACATGAGGAT

[0303] ATTAATCTGCAAGAGATTATTAGCGCAGCCGGTAAAGAACTGTCAGAAGCATTT AAACAGAAAACCAGCGAAATTCTGTCACATGCACATGCAGCACTGGATCAGCCG CTGCCGACCACCCTGAAAAAACAAGAAGAAAAAGAAATCCTGAAAAGCCAGCT

[0304] GGATAGCCTGCTGGGTCTGTATCATCTGCTGGACTGGTTTGCAGTTGATGAAAGC

[0305] AATGAAGTTGATCCGGAATTTAGCGCACGTCTGACCGGCATTAAACTGGAAATG

[0306] GAACCGAGCCTGAGCTTTTATAACAAAGCCCGTAATTATGCCACCAAAAAACCG

[0307] TATAGCGTCGAAAAATTCAAACTGAACTTTCAGCGTCCGACCCTGGCAAGCGGTT

[0308] GGGATGTTAATAAAGAAAAAAACAACGGTGCCATCCTGTTCGTGAAAAATGGCC

[0309] TGTATTATCTGGGTATTATGCCGAAACAGAAAGGTCGTTATAAAGCGCTGAGCTT

[0310] TGAACCGACGGAAAAAACCAGTGAAGGTTTTGATAAAATGTACTACGACTATTTT

[0311] CCGGATGCAGCCAAAATGATTCCGAAATGTAGCACCCAGCTGAAAGCAGTTACC

[0312] GCACATTTTCAGACCCATACCACCCCGATTCTGCTGAGCAATAACTTTATTGAAC

[0313] CGCTGGAAATCACCAAAGAGATCTACGATCTGAATAACCCGGAAAAAGAGCCGA

[0314] AAAAATTCCAGACCGCATATGCAAAAAAAACCGGTGATCAGAAAGGTTATCGTG

[0315] AAGCGCTGTGTAAATGGATTGATTTCACCCGTGATTTTCTGAGCAAATACACCAA

[0316] AACCACCAGTATCGATCTGAGCAGCCTGCGTCCGAGCAGCCAGTATAAAGATCT

[0317] GGGCGAATATTATGCAGAACTGAATCCGCTGCTGTATCATATTAGCTTTCAGCGT

[0318] ATTGCCGAGAAAGAAATCATGGACGCAGTTGAAACCGGTAAACTGTACCTGTTC

[0319] CAGATCTACAATAAAGATTTTGCCAAAGGCCATCATGGCAAACCGAATCTGCAT

[0320] ACC CTGTATTGGACC GGTCTGTTTAGCC CTGAAAATCTGGC AAAAAC CTC GATTA

[0321] AACTGAATGGTCAGGCGGAACTGTTTTATCGTCCGAAAAGCCGTATGAAACGTA

[0322] TGGCAGCTCGTCTGGGTGAAAAAATGCTGAACAAAAAACTGAAAGACCAGAAA

[0323] ACCCCGATCCCGGATACACTGTATCAAGAACTGTATGATTATGTGAACCATCGTC

[0324] TGAGCCATGATCTGAGTGATGAAGCACGTGCCCTGCTGCCGAATGTTATTACCAA

[0325] AGAAGTTAGCCACGAGATCATTAAAGATCGTCGTTTTACCAGCGACAAATTCCTG

[0326] TTTCATGTGCCGATTACCCTGAATTATCAGGCAGCAAATAGCCCGAGCAAATTTA

[0327] ACCAGCGTGTTAATGCATATCTGAAAGAACATCCAGAAACGCCGATTATTGGTAT

[0328] TGATCGTGGTGAACGTAACCTGATTTATATCACCGTTATTGATAGCACCGGCAAA

[0329] ATCCTGGAACAGCGTAGCCTGAATACCATTCAGCAGTTTGATTACCAGAAAAAA

[0330] CTGGATAATCGCGAGAAAGAACGTGTTGCAGCACGTCAGGCATGGTCAGTTGTT

[0331] GGTACAATTAAAGACCTGAAACAGGGTTATCTGAGCCAGGTTATTCATGAAATTG

[0332] TGGATCTGATGATTCACTATCAGGCCGTTGTTGTGCTGGAAAACCTGAATTTTGG

[0333] CTTTAAAAGCAAACGTACCGGCATTGCAGAAAAAGCAGTTTATCAGCAGTTCGA

[0334] GAAAATGCTGATTGACAAACTGAATTGCCTGGTGCTGAAAGATTATCCGGCTGA

[0335] AAAAGTTGGTGGTGTTCTGAATCCGTATCAGCTGACCGATCAGTTTACCAGCTTT

[0336] GCAAAAATGGGCACCCAGAGCGGATTTCTGTTTTATGTTCCGGCACCGTATACGA GCAAAATTGATCCGCTGACCGGTTTTGTTGATCCGTTTGTTTGGAAAACCATCAA AAACCATGAAAGCCGCAAACATTTTCTGGAAGGTTTCGATTTTCTGCATTACGAC GTTAAAACGGGTGATTTCATCCTGCACTTTAAAATGAATCGCAATCTGAGTTTTC AGCGTGGCCTGCCTGGTTTTATGCCTGCATGGGATATTGTGTTTGAGAAAAACGA AACACAGTTCGATGCAAAAGGCACCCCGTTTATTGCAGGTAAACGTATTGTTCCG GTGATTGAAAATCATCGTTTCACCGGTCGTTATCGCGATCTGTATCCGGCAAATG AACTGATCGCACTGCTGGAAGAGAAAGGTATTGTTTTTCGTGATGGCTCAAACAT TCTGCCGAAACTGCTGGAAAATGATGATAGCCATGCAATTGATACCATGGTTGCA CTGATTCGTAGCGTTCTGCAGATGCGTAATAGCAATGCAGCAACCGGTGAAGATT ACATTAATAGTCCGGTTCGTGATCTGAATGGTGTTTGTTTTGATAGCCGTTTTCAG AATCCGGAATGGCCGATGGATGCAGATGCAAATGGTGCATATCATATTGCACTG AAAGGACAGCTGCTGCTGAACCACCTGAAAGAAAGCAAAGATCTGAAACTGCAA AACGGCATTAGCAATCAGGATTGGCTGGCATATATCCAAGAACTGCGTAACGGT CGTAGCAGTGATGATGAAGCAACCGCAGATAGCCAGCATGCAGCACCGCCTAAA AAGAAACGTAAAGTT [SEQ ID NO: 121]

[0337] Exemplary AsCasl2a variant 5 amino acid sequence:

[0338] MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPI IDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYF IGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTT YFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVK KAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAI QKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENV LETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKS AKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQE EKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARN YATKKPYSVEKFKLNFQRPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGR YKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNN FIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYT KTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIY NKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHR LGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHE IIKDRRFTSDKFLFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYI TVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLS QVIHEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLK DYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVW KTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFE KNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNI LPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQN PEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQELRNGRS SDDEATADSQHAAPPKKKRKV [SEQ ID NO: 76]

[0339] Exemplary AsCas 12a variant 6 amino acid sequence:

[0340] MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPI IDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYF IGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTT YFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVK KAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAI QKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENV LETAEAEFNELNSIDLTHIFISHKKLET1SSALCDHWDTLRNALYERR1SELTGKITKS AKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQE EKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARN YATKKPYSVEKFKLNFQRPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGR YKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNN FIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYT KTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIY NKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHR LGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHE IIKDRRFTSDKFLFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYI TVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLS QVIHEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLK DYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVW KTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFE KNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNI LPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQN PEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQELRNGRS SDDEATADSQHAAPPKKKRKV GGSGGSGGSGGSGGSGGSGGSGGSLEHHHHHH [SEQ ID NO: 77] Exemplary AsCas 12a variant 7 amino acid sequence:

[0341] MGRDPGKPIPNPLLGLDSTAPKKKRKVGIHGVPAATQFEGFTNLYQVSKTLR FELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYADQCLQLVQLDWEN LSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLF KAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDISTAIP HRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQLL TQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQI

[0342] LSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHK KLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAA GKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWF AVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTL ASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYY DYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPK

[0343] KFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGE YYAELNPLLYHISFQR1AEKE1MDAVETGKLYLFQ1YNKDFAKGHHGKPNLHTLYW TGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTPIPDT LYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFFHVPITLNY QAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQF DYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLE

[0344] NLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQ FTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLH YDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVP VIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIR SVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQ LLLNHLKESKDLKLQNGISNQDWLAYIQELRNPKKKRKVKLAAALEHHHHHH

[0345] [SEQ ID NO: 78]

[0346] Exemplary AsCas 12a variant 8 amino acid sequence:

[0347] MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPI IDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYF IGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTT YFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVK KAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAI QKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENV LETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKS AKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQE EKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARN YATKKPYSVEKFKLNFQRPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGR YKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNN FIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYT KTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIY NKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAAR LGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHE IIKDRRFTSDKFLFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYI TVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLS QVIHEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLK DYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVW KTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFE KNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNI LPKLLENDDSHA1DTMVAL1RSVLQMRNSNAATGEDY1NSPVRDLNGVCFDSRFQN PEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQELRNGGS

[0348] PAAKRVKLDGGSPAAKRVKLD [SEQ ID NO: 431]

[0349] Exemplary As Cas12a variant 9 amino acid sequence:

[0350] MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPI IDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYF IGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTT YFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVK KAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAI QKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENV LETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKS AKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQE EKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARN YATKKPYSVEKFKLNFQRPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGR YKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNN FIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYT KTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIY NKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAAR LGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHE IIKDRRFTSDKFLFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYI TVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLS QVIHEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLK DYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVW KTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFE KNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNI LPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQN PEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQELRN

[0351] [SEQ ID NO: 432]

[0352] Exemplary AsCas 12a wild-type amino acid sequence:

[0353] MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPI

[0354] IDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYF IGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTT YFSGFYENRKNVFSAEDISTA1PHR1VQDNFPKFKENCH1FTRLITAVPSLREHFENVK KAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAI

[0355] QKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENV LETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKS

[0356] AKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQE EKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARN YATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGR YKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNN FIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYT KTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIY NKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHR LGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHE IIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYI

[0357] TVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLS QVIHEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLK

[0358] DYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVW KTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFE KNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNI LPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQN PEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQELRN [SEQ ID NO: 79]

[0359] Exemplary AsCasl2a-MHFRR amino acid sequence:

[0360] MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPII DRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFI GRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTY FSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKK AIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQK NDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLET AEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEK VQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEIL KSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNYATKK PYSVEKFKLNFQRPTLARGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSF EPTEKTSEGFDKMYYDYFPDAAKMIPRCSTQLKAVTAHFQTHTTPILLSNNFIEPLEIT KEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKW1DFTRDFLSKYTKTTS1DLS SLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGH HGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAARLGEKMLNK KLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSD KFLFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKIL EQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMI HYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVL NPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFL EGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFI AGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDT MVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHI ALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQELRNGGSPAAKRVKLDGGSPAAK RVKLD [SEQ ID NO: 423]

[0361] Exemplary AsCasl2a-MHFRR amino acid sequence:

[0362] MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPII DRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFI GRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTY FSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKK AIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQK NDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLET AEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEK VQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEIL KSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNYATKK PYSVEKFKLNFQRPTLARGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSF EPTEKTSEGFDKMYYDYFPDAAKMIPRCSTQLKAVTAHFQTHTTPILLSNNFIEPLEIT KEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSTDLS SLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGH HGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAARLGEKMLNK KLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSD KFLFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKIL

[0363] EQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMI HYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVL NPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFL EGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPF1 AGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDT MVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHI ALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQELRNGRSSDDEATADSQHAAPPK KKRKV [SEQ ID NO: 408]

[0364] Exemplary AsCasl2a-MHFRVR amino acid sequence:

[0365] MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPII DRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFI GRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTY FSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKK AIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQK NDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLET AEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEK VQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEIL KSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNYATKK PYSVEKFKLNFQRPTLARGWDVNVEKNRGAILFVKNGLYYLGIMPKQKGRYKALSF EPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEIT KEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLS SLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGH HGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAARLGEKMLNK KLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSD KFLFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKIL EQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMI HYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVL NPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFL EGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFI AGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDT MVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHI ALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQELRNGGSPAAKRVKLDGGSPAAK RVKLD [SEQ ID NO: 424]

[0366] Exemplary AsCasl2a-MHFRVR amino acid sequence:

[0367] MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPI IDR1YKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNA1HDYF IGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTT YFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVK KAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAI QKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENV LETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKS AKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQE EKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARN YATKKPYSVEKFKLNFQRPTLARGWDVNVEKNRGAILFVKNGLYYLGIMPKQKGR YKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNN FIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYT KTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIY NKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAAR LGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHE IIKDRRFTSDKFLFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYI TVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLS QVIHEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLK DYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVW KTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFE KNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNI LPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQN PEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQELRNGRS SDDEATADSQHAAPPKKKRKV [SEQ ID NO: 409]

[0368] Exemplary AsCasl2a-MHF amino acid sequence:

[0369] MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPII DRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFI GRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTY FSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKK AIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQK NDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLET AEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEK VQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEIL KSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNYATKK PYSVEKFKLNFQRPTLASGWDVNKEKNNGA1LFVKNGLYYLG1MPKQKGRYKALSF EPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEIT KEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLS SLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGH HGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAARLGEKMLNK KLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSD KFLFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKIL EQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMI HYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVL NPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFL EGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFI AGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDT MVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHI ALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQELRNGRSSDDEATADSQHAAPPK KKRKV [SEQ ID NO: 430]

[0370] Exemplary AsCasl2a-MF mRNA sequence:

[0371] AGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCA UGACACAGUUCGAGGGCUUUACCAACCUGUAUCAGGUGAGCAAGACACUGCGG UUUGAGCUGAUCCCACAGGGCAAGACCCUGAAGCACAUCCAGGAGCAGGGCUU CAUCGAGGAGGACAAGGCCCGCAAUGAUCACUACAAGGAGCUGAAGCCCAUCA

[0372] UCGAUCGGAUCUACAAGACCUAUGCCGACCAGUGCCUGCAGCUGGUGCAGCUG

[0373] GAUUGGGAGAACCUGAGCGCCGCCAUCGACUCCUAUAGAAAGGAGAAAACCGA

[0374] GGAGACAAGGAACGCCCUGAUCGAGGAGCAGGCCACAUAUCGCAAUGCCAUCC

[0375] ACGACUACUUCAUCGGCCGGACAGACAACCUGACCGAUGCCAUCAAUAAGAGA

[0376] CACGCCGAGAUCUACAAGGGCCUGUUCAAGGCCGAGCUGUUUAAUGGCAAGGU

[0377] GCUGAAGCAGCUGGGCACCGUGACCACAACCGAGCACGAGAACGCCCUGCUGC

[0378] GGAGCUUCGACAAGUUUACAACCUACUUCUCCGGCUUUUAUGAGAACAGGAA

[0379] GAACGUGUUCAGCGCCGAGGAUAUCAGCACAGCCAUCCCACACCGCAUCGUGC

[0380] AGGACAACUUCCCCAAGUUUAAGGAGAAUUGUCACAUCUUCACACGCCUGAUC

[0381] ACCGCCGUGCCCAGCCUGCGGGAGCACUUUGAGAACGUGAAGAAGGCCAUCGG

[0382] CAUCUUCGUGAGCACCUCCAUCGAGGAGGUGUUUUCCUUCCCUUUUUAUAACC

[0383] AGCUGCUGACACAGACCCAGAUCGACCUGUAUAACCAGCUGCUGGGAGGAAUC

[0384] UCUCGGGAGGCAGGCACCGAGAAGAUCAAGGGCCUGAACGAGGUGCUGAAUC

[0385] UGGCCAUCCAGAAGAAUGAUGAGACAGCCCACAUCAUCGCCUCCCUGCCACAC

[0386] AGAUUCAUCCCCCUGUUUAAGCAGAUCCUGUCCGAUAGGAACACCCUGUCUUU

[0387] CAUCCUGGAGGAGUUUAAGAGCGACGAGGAAGUGAUCCAGUCCUUCUGCAAG

[0388] UACAAGACACUGCUGAGAAACGAGAACGUGCUGGAGACAGCCGAGGCCCUGUU

[0389] UAACGAGCUGAACAGCAUCGACCUGACACACAUCUUCAUCAGCCACAAGAAGC

[0390] UGGAGACAAUCAGCAGCGCCCUGUGCGACCACUGGGAUACACUGAGGAAUGCC

[0391] CUGUAUGAGCGGAGAAUCUCCGAGCUGACAGGCAAGAUCACCAAGUCUGCCAA

[0392] GGAGAAGGUGCAGCGCAGCCUGAAGCACGAGGAUAUCAACCUGCAGGAGAUC

[0393] AUCUCUGCCGCAGGCAAGGAGCUGAGCGAGGCCUUCAAGCAGAAAACCAGCGA

[0394] GAUCCUGUCCCACGCACACGCCGCCCUGGAUCAGCCACUGCCUACAACCCUGA

[0395] AGAAGCAGGAGGAGAAGGAGAUCCUGAAGUCUCAGCUGGACAGCCUGCUGGG

[0396] CCUGUACCACCUGCUGGACUGGUUUGCCGUGGAUGAGUCCAACGAGGUGGACC

[0397] CCGAGUUCUCUGCCCGGCUGACCGGCAUCAAGCUGGAGAUGGAGCCUUCUCUG

[0398] AGCUUCUACAACAAGGCCAGAAAUUAUGCCACCAAGAAGCCCUACUCCGUGGA

[0399] GAAGUUCAAGCUGAACUUUCAGCGGCCUACACUGGCCUCUGGCUGGGACGUGA

[0400] AUAAGGAGAAGAACAAUGGCGCCAUCCUGUUUGUGAAGAACGGCCUGUACUA

[0401] UCUGGGCAUCAUGCCAAAGCAGAAGGGCAGGUAUAAGGCCCUGAGCUUCGAGC

[0402] CCACAGAGAAAACCAGCGAGGGCUUUGAUAAGAUGUACUAUGACUACUUCCCU

[0403] GAUGCCGCCAAGAUGAUCCCAAAGUGCAGCACCCAGCUGAAGGCCGUGACAGC

[0404] CCACUUUCAGACCCACACAACCCCCAUCCUGCUGUCCAACAAUUUCAUCGAGC CUCUGGAGAUCACAAAGGAGAUCUACGACCUGAACAAUCCUGAGAAGGAGCCA

[0405] AAGAAGUUUCAGACAGCCUACGCCAAGAAAACCGGCGACCAGAAGGGCUACAG

[0406] AGAGGCCCUGUGCAAGUGGAUCGACUUCACAAGGGAUUUUCUGUCCAAGUAU

[0407] ACCAAGACAACCUCUAUCGAUCUGUCUAGCCUGCGGCCAUCCUCUCAGUAUAA

[0408] GGACCUGGGCGAGUACUAUGCCGAGCUGAAUCCCCUGCUGUACCACAUCAGCU

[0409] UCCAGAGAAUCGCCGAGAAGGAGAUCAUGGAUGCCGUGGAGACAGGCAAGCU

[0410] GUACCUGUUCCAGAUCUAUAACAAGGACUUUGCCAAGGGCCACCACGGCAAGC

[0411] CUAAUCUGCACACACUGUAUUGGACCGGCCUGUUUUCUCCAGAGAACCUGGCC

[0412] AAGACAAGCAUCAAGCUGAAUGGCCAGGCCGAGCUGUUCUACCGCCCUAAGUC

[0413] CAGGAUGAAGAGGAUGGCACACCGGCUGGGAGAGAAGAUGCUGAACAAGAAG

[0414] CUGAAGGAUCAGAAAACCCCAAUCCCCGACACCCUGUACCAGGAGCUGUACGA

[0415] CUAUGUGAAUCACAGACUGUCCCACGACCUGUCUGAUGAGGCCAGGGCCCUGC

[0416] UGCCCAACGUGAUCACCAAGGAGGUGUCUCACGAGAUCAUCAAGGAUAGGCGC

[0417] UUUACCAGCGACAAGUUCUUGUUCCACGUGCCUAUCACACUGAACUAUCAGGC

[0418] CGCCAAUUCCCCAUCUAAGUUCAACCAGAGGGUGAAUGCCUACCUGAAGGAGC

[0419] ACCCCGAGACACCUAUCAUCGGCAUCGAUCGGGGCGAGAGAAACCUGAUCUAU

[0420] AUCACAGUGAUCGACUCCACCGGCAAGAUCCUGGAGCAGCGGAGCCUGAACAC

[0421] CAUCCAGCAGUUUGAUUACCAGAAGAAGCUGGACAACAGGGAGAAGGAGAGG

[0422] GUGGCAGCAAGGCAGGCCUGGUCUGUGGUGGGCACAAUCAAGGAUCUGAAGC

[0423] AGGGCUAUCUGAGCCAGGUCAUCCACGAGAUCGUGGACCUGAUGAUCCACUAC

[0424] CAGGCCGUGGUGGUGCUGGAGAACCUGAAUUUCGGCUUUAAGAGCAAGAGGA

[0425] CCGGCAUCGCCGAGAAGGCCGUGUACCAGCAGUUCGAGAAGAUGCUGAUCGAU

[0426] AAGCUGAAUUGCCUGGUGCUGAAGGACUAUCCAGCAGAGAAAGUGGGAGGCG

[0427] UGCUGAACCCAUACCAGCUGACAGACCAGUUCACCUCCUUUGCCAAGAUGGGC

[0428] ACCCAGUCUGGCUUCCUGUUUUACGUGCCUGCCCCAUAUACAUCUAAGAUCGA

[0429] UCCCCUGACCGGCUUCGUGGACCCCUUCGUGUGGAAAACCAUCAAGAAUCACG

[0430] AGAGCCGCAAGCACUUCCUGGAGGGCUUCGACUUUCUGCACUACGACGUGAAA

[0431] ACCGGCGACUUCAUCCUGCACUUUAAGAUGAACAGAAAUCUGUCCUUCCAGAG

[0432] GGGCCUGCCCGGCUUUAUGCCUGCAUGGGAUAUCGUGUUCGAGAAGAACGAG

[0433] ACACAGUUUGACGCCAAGGGCACCCCUUUCAUCGCCGGCAAGAGAAUCGUGCC

[0434] AGUGAUCGAGAAUCACAGAUUCACCGGCAGAUACCGGGACCUGUAUCCUGCCA

[0435] ACGAGCUGAUCGCCCUGCUGGAGGAGAAGGGCAUCGUGUUCAGGGAUGGCUCC

[0436] AACAUCCUGCCAAAGCUGCUGGAGAAUGACGAUUCUCACGCCAUCGACACCAU

[0437] GGUGGCCCUGAUCCGCAGCGUGCUGCAGAUGCGGAACUCCAAUGCCGCCACAG GCGAGGACUAUAUCAACAGCCCCGUGCGCGAUCUGAAUGGCGUGUGCUUCGAC UCCCGGUUUCAGAACCCAGAGUGGCCCAUGGACGCCGAUGCCAAUGGCGCCUA CCACAUCGCCCUGAAGGGCCAGCUGCUGCUGAAUCACCUGAAGGAGAGCAAGG AUCUGAAGCUGCAGAACGGCAUCUCCAAUCAGGACUGGCUGGCCUACAUCCAG GAGCUGCGCAACGGUCGUAGCAGUGAUGAUGAAGCAACCGCAGAUAGCCAGCA UGCAGCACCCAAGAAGAAGAGGAAAGUCUAAUAGUGAAUGGUUUAUAUUGCG GCCGCUUAAUUAAGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCU UCUCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUAGGAAG AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAA [SEQ ID NO: 429]

[0438] In some embodiments, an RNA-guided nuclease has at least 80%, at least 85%, at least 86%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%. at least 99.7%. at least 99.8%, or at least 99.9% sequence identity relative to a wildtype RNA-guided nuclease and / or an RNA-guided nuclease disclosed herein (e.g, an RNA- guided nuclease comprising an amino acid sequence selected from the group consisting of SEQ iD NOs: 71-79, 423, 424, and 430-432). In some embodiments, an RNA-guided nuclease has 1, 2, 3, 4. 5, 6, 7. 8, 9, 10, 11, 12, 13, 14, 15. 16. 17. 18. 19, or 20 mutations relative to a wildtype RNA-guided nuclease and / or an RNA-guided nuclease disclosed herein (e.g, an RNA- guided nuclease comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 71-79, 423, 424, and 430-432). In some embodiments, an RNA-guided nuclease has less than 2. 3, 4, 5, 6, 7, 8, 9, 10. 11. 12. 13, 14, 15, 16, 17, 18, 19, or 20 mutations relative to a wild-type RNA-guided nuclease and / or an RNA-guided nuclease disclosed herein (e.g, an RNA-guided nuclease comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 71-79, 423, 424, and 430-432).

[0439] 5.4 Nucleic acids encoding RNA-guided nucleases

[0440] Nucleic acids encoding RNA-guided nucleases, e.g.. Cas9, Casl2a or functional fragments thereof, are provided herein. Exemplar}' nucleic acids encoding RNA-guided nucleases have been described previously (see, e.g , Cong 2013; Wang 2013; Mali 2013; Jinek 2012).

[0441] In some cases, a nucleic acid encoding an RNA-guided nuclease can be a synthetic nucleic acid sequence. For example, the synthetic nucleic acid molecule can be chemically modified. In certain embodiments, the nucleic acid encoding the RNA-guided nuclease is an RNA. In certain embodiments, the nucleic acid encoding the RNA-guided nuclease is an mRNA. In certain embodiments, an mRNA encoding an RNA-guided nuclease will have one or more (e.g., all) of the following properties: it can be capped; polyadenylated; and substituted with 5 -methylcytidine and / or pseudouridine.

[0442] Synthetic nucleic acid sequences can also be codon optimized, e.g., at least one noncommon codon or less-common codon has been replaced by a common codon. For example, the synthetic nucleic acid can direct the synthesis of an optimized messenger mRNA, e.g , optimized for expression in a mammalian expression system, e.g, described herein. Examples of codon optimized Cas9 coding sequences are presented in Cotta-Ramusino.

[0443] In addition, or alternatively, a nucleic acid encoding an RNA-guided nuclease can comprise a nucleic acid encoding a nuclear localization sequence (NLS). Nuclear localization sequences are known in the art and examples include but are not limited to the NLS sequences fused to the RNA-guided nuclease sequence as indicated in SEQ ID NO: 71, presented herein.

[0444] Some non-limiting exemplary RNA sequences encoding the RNA-guided nuclease are as follows: mRNA #l

[0445] (5’ modification is CleanCap® AG; 3'Tail Modification is a 79A poly A sequence) AGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCA

[0446] UGACACAGUUCGAGGGCUUUACCAACCUGUAUCAGGUGAGCAAGACACUGCGG UUUGAGCUGAUCCCACAGGGCAAGACCCUGAAGCACAUCCAGGAGCAGGGCUU CAUCGAGGAGGACAAGGCCCGCAAUGAUCACUACAAGGAGCUGAAGCCCAUCA UCGAUCGGAUCUACAAGACCUAUGCCGACCAGUGCCUGCAGCUGGUGCAGCUG GAUUGGGAGAACCUGAGCGCCGCCAUCGACUCCUAUAGAAAGGAGAAAACCGA GGAGACAAGGAACGCCCUGAUCGAGGAGCAGGCCACAUAUCGCAAUGCCAUCC ACGACUACUUCAUCGGCCGGACAGACAACCUGACCGAUGCCAUCAAUAAGAGA CACGCCGAGAUCUACAAGGGCCUGUUCAAGGCCGAGCUGUUUAAUGGCAAGGU GCUGAAGCAGCUGGGCACCGUGACCACAACCGAGCACGAGAACGCCCUGCUGC GGAGCUUCGACAAGUUUACAACCUACUUCUCCGGCUUUUAUGAGAACAGGAA GAACGUGUUCAGCGCCGAGGAUAUCAGCACAGCCAUCCCACACCGCAUCGUGC AGGACAACUUCCCCAAGUUUAAGGAGAAUUGUCACAUCUUCACACGCCUGAUC ACCGCCGUGCCCAGCCUGCGGGAGCACUUUGAGAACGUGAAGAAGGCCAUCGG CAUCUUCGUGAGCACCUCCAUCGAGGAGGUGUUUUCCUUCCCUUUUUAUAACC AGCUGCUGACACAGACCCAGAUCGACCUGUAUAACCAGCUGCUGGGAGGAAUC

[0447] UCUCGGGAGGCAGGCACCGAGAAGAUCAAGGGCCUGAACGAGGUGCUGAAUC

[0448] UGGCCAUCCAGAAGAAUGAUGAGACAGCCCACAUCAUCGCCUCCCUGCCACAC

[0449] AGAUUCAUCCCCCUGUUUAAGCAGAUCCUGUCCGAUAGGAACACCCUGUCUUU

[0450] CAUCCUGGAGGAGUUUAAGAGCGACGAGGAAGUGAUCCAGUCCUUCUGCAAG

[0451] UACAAGACACUGCUGAGAAACGAGAACGUGCUGGAGACAGCCGAGGCCCUGUU

[0452] UAACGAGCUGAACAGCAUCGACCUGACACACAUCUUCAUCAGCCACAAGAAGC

[0453] UGGAGACAAUCAGCAGCGCCCUGUGCGACCACUGGGAUACACUGAGGAAUGCC

[0454] CUGUAUGAGCGGAGAAUCUCCGAGCUGACAGGCAAGAUCACCAAGUCUGCCAA

[0455] GGAGAAGGUGCAGCGCAGCCUGAAGCACGAGGAUAUCAACCUGCAGGAGAUC

[0456] AUCUCUGCCGCAGGCAAGGAGCUGAGCGAGGCCUUCAAGCAGAAAACCAGCGA

[0457] GAUCCUGUCCCACGCACACGCCGCCCUGGAUCAGCCACUGCCUACAACCCUGA

[0458] AGAAGCAGGAGGAGAAGGAGAUCCUGAAGUCUCAGCUGGACAGCCUGCUGGG

[0459] CCUGUACCACCUGCUGGACUGGUUUGCCGUGGAUGAGUCCAACGAGGUGGACC

[0460] CCGAGUUCUCUGCCCGGCUGACCGGCAUCAAGCUGGAGAUGGAGCCUUCUCUG

[0461] AGCUUCUACAACAAGGCCAGAAAUUAUGCCACCAAGAAGCCCUACLICCGLIGGA

[0462] GAAGUUCAAGCUGAACUUUCAGCGGCCUACACUGGCCUCUGGCUGGGACGUGA

[0463] AUAAGGAGAAGAACAAUGGCGCCAUCCUGUUUGUGAAGAACGGCCUGUACUA

[0464] UCUGGGCAUCAUGCCAAAGCAGAAGGGCAGGUAUAAGGCCCUGAGCUUCGAGC

[0465] CCACAGAGAAAACCAGCGAGGGCUUUGAUAAGAUGUACUAUGACUACUUCCCU

[0466] GAUGCCGCCAAGAUGAUCCCAAAGUGCAGCACCCAGCUGAAGGCCGUGACAGC

[0467] CCACUUUCAGACCCACACAACCCCCAUCCUGCUGUCCAACAAUUUCAUCGAGC

[0468] CUCUGGAGAUCACAAAGGAGAUCUACGACCUGAACAAUCCUGAGAAGGAGCCA

[0469] AAGAAGUUUCAGACAGCCUACGCCAAGAAAACCGGCGACCAGAAGGGCUACAG

[0470] AGAGGCCCUGUGCAAGUGGAUCGACUUCACAAGGGAUUUUCUGUCCAAGUAU

[0471] ACCAAGACAACCUCUAUCGAUCUGUCUAGCCUGCGGCCAUCCUCUCAGUAUAA

[0472] GGACCUGGGCGAGUACUAUGCCGAGCUGAAUCCCCUGCUGUACCACAUCAGCU

[0473] UCCAGAGAAUCGCCGAGAAGGAGAUCAUGGAUGCCGUGGAGACAGGCAAGCU

[0474] GUACCUGUUCCAGAUCUAUAACAAGGACUUUGCCAAGGGCCACCACGGCAAGC

[0475] CUAAUCUGCACACACUGUAUUGGACCGGCCUGUUUUCUCCAGAGAACCUGGCC

[0476] AAGACAAGCAUCAAGCUGAAUGGCCAGGCCGAGCUGUUCUACCGCCCUAAGUC

[0477] CAGGAUGAAGAGGAUGGCAGCCCGGCUGGGAGAGAAGAUGCUGAACAAGAAG

[0478] CUGAAGGAUCAGAAAACCCCAAUCCCCGACACCCUGUACCAGGAGCUGUACGA

[0479] CUAUGUGAAUCACAGACUGUCCCACGACCUGUCUGAUGAGGCCAGGGCCCUGC UGCCCAACGUGAUCACCAAGGAGGUGUCUCACGAGAUCAUCAAGGAUAGGCGC

[0480] UUUACCAGCGACAAGUUCUUGUUCCACGUGCCUAUCACACUGAACUAUCAGGC

[0481] CGCCAAUUCCCCAUCUAAGUUCAACCAGAGGGUGAAUGCCUACCUGAAGGAGC

[0482] ACCCCGAGACACCUAUCAUCGGCAUCGAUCGGGGCGAGAGAAACCUGAUCUAU

[0483] AUCACAGUGAUCGACUCCACCGGCAAGAUCCUGGAGCAGCGGAGCCUGAACAC

[0484] CAUCCAGCAGUUUGAUUACCAGAAGAAGCUGGACAACAGGGAGAAGGAGAGG

[0485] GUGGCAGCAAGGCAGGCCUGGUCUGUGGUGGGCACAAUCAAGGAUCUGAAGC

[0486] AGGGCUAUCUGAGCCAGGUCAUCCACGAGAUCGUGGACCUGAUGAUCCACUAC

[0487] CAGGCCGUGGUGGUGCUGGAGAACCUGAAUUUCGGCUUUAAGAGCAAGAGGA

[0488] CCGGCAUCGCCGAGAAGGCCGUGUACCAGCAGUUCGAGAAGAUGCUGAUCGAU

[0489] AAGCUGAAUUGCCUGGUGCUGAAGGACUAUCCAGCAGAGAAAGUGGGAGGCG

[0490] UGCUGAACCCAUACCAGCUGACAGACCAGUUCACCUCCUUUGCCAAGAUGGGC

[0491] ACCCAGUCUGGCUUCCUGUUUUACGUGCCUGCCCCAUAUACAUCUAAGAUCGA

[0492] UCCCCUGACCGGCUUCGUGGACCCCUUCGUGUGGAAAACCAUCAAGAAUCACG

[0493] AGAGCCGCAAGCACUUCCUGGAGGGCUUCGACUUUCUGCACUACGACGUGAAA

[0494] ACCGGCGACUUCAUCCUGCACUUUAAGAUGAACAGAAAUCUGUCCUUCCAGAG

[0495] GGGCCUGCCCGGCUUUAUGCCUGCAUGGGAUAUCGUGUUCGAGAAGAACGAG

[0496] ACACAGUUUGACGCCAAGGGCACCCCUUUCAUCGCCGGCAAGAGAAUCGUGCC

[0497] AGUGAUCGAGAAUCACAGAUUCACCGGCAGAUACCGGGACCUGUAUCCUGCCA

[0498] ACGAGCUGAUCGCCCUGCUGGAGGAGAAGGGCAUCGUGUUCAGGGAUGGCUCC

[0499] AACAUCCUGCCAAAGCUGCUGGAGAAUGACGAUUCUCACGCCAUCGACACCAU

[0500] GGUGGCCCUGAUCCGCAGCGUGCUGCAGAUGCGGAACUCCAAUGCCGCCACAG

[0501] GCGAGGACUAUAUCAACAGCCCCGUGCGCGAUCUGAAUGGCGUGUGCUUCGAC

[0502] UCCCGGUUUCAGAACCCAGAGUGGCCCAUGGACGCCGAUGCCAAUGGCGCCUA

[0503] CCACAUCGCCCUGAAGGGCCAGCUGCUGCUGAAUCACCUGAAGGAGAGCAAGG

[0504] AUCUGAAGCUGCAGAACGGCAUCUCCAAUCAGGACUGGCUGGCCUACAUCCAG

[0505] GAGCUGCGCAACGGUCGUAGCAGUGAUGAUGAAGCAACCGCAGAUAGCCAGCA

[0506] UGCAGCACCGCCCAAGAAGAAGAGGAAAGUCUAAUAGUGAAUGGUUUAUAUU

[0507] GCGGCCGCUUAAUUAAGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCU

[0508] UCUUCUCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUAGG

[0509] AAGUCUAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0510] AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA [SEQ ID NO: 93] mRNA #2 (5’ Modification = CleanCap® AG 3’0me; Uridine Modifications = Nl-Methyl- Pseudo UTP; 3' Tail Modification = 79A tail)

[0511] AGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCA

[0512] UGACACAGUUCGAGGGCUUUACCAACCUGUAUCAGGUGAGCAAGACACUGCGG UUUGAGCUGAUCCCACAGGGCAAGACCCUGAAGCACAUCCAGGAGCAGGGCUU CAUCGAGGAGGACAAGGCCCGCAAUGAUCACUACAAGGAGCUGAAGCCCAUCA UCGAUCGGAUCUACAAGACCUAUGCCGACCAGUGCCUGCAGCUGGUGCAGCUG GAUUGGGAGAACCUGAGCGCCGCCAUCGACUCCUAUAGAAAGGAGAAAACCGA GGAGACAAGGAACGCCCUGAUCGAGGAGCAGGCCACAUAUCGCAAUGCCAUCC ACGACUACUUCAUCGGCCGGACAGACAACCUGACCGAUGCCAUCAAUAAGAGA CACGCCGAGAUCUACAAGGGCCUGUUCAAGGCCGAGCUGUUUAAUGGCAAGGU GCUGAAGCAGCUGGGCACCGUGACCACAACCGAGCACGAGAACGCCCUGCUGC GGAGCUUCGACAAGUUUACAACCUACUUCUCCGGCUUUUAUGAGAACAGGAA GAACGUGUUCAGCGCCGAGGAUAUCAGCACAGCCAUCCCACACCGCAUCGUGC AGGACAACUUCCCCAAGUUUAAGGAGAAUUGUCACAUCUUCACACGCCUGAUC ACCGCCGUGCCCAGCCUGCGGGAGCACUUUGAGAACGUGAAGAAGGCCAUCGG CAUCUUCGUGAGCACCUCCAUCGAGGAGGUGUUUUCCUUCCCUUUUUAUAACC AGCUGCUGACACAGACCCAGAUCGACCUGUAUAACCAGCUGCUGGGAGGAAUC UCUCGGGAGGCAGGCACCGAGAAGAUCAAGGGCCUGAACGAGGUGCUGAAUC UGGCCAUCCAGAAGAAUGAUGAGACAGCCCACAUCAUCGCCUCCCUGCCACAC AGAUUCAUCCCCCUGUUUAAGCAGAUCCUGUCCGAUAGGAACACCCUGUCUUU CAUCCUGGAGGAGUUUAAGAGCGACGAGGAAGUGAUCCAGUCCUUCUGCAAG UACAAGACACUGCUGAGAAACGAGAACGUGCUGGAGACAGCCGAGGCCCUGUU UAACGAGCUGAACAGCAUCGACCUGACACACAUCUUCAUCAGCCACAAGAAGC UGGAGACAAUCAGCAGCGCCCUGUGCGACCACUGGGAUACACUGAGGAAUGCC

[0513] CUGUAUGAGCGGAGAAUCUCCGAGCUGACAGGCAAGAUCACCAAGUCUGCCAA GGAGAAGGUGCAGCGCAGCCUGAAGCACGAGGAUAUCAACCUGCAGGAGAUC AUCUCUGCCGCAGGCAAGGAGCUGAGCGAGGCCUUCAAGCAGAAAACCAGCGA GAUCCUGUCCCACGCACACGCCGCCCUGGAUCAGCCACUGCCUACAACCCUGA AGAAGCAGGAGGAGAAGGAGAUCCUGAAGUCUCAGCUGGACAGCCUGCUGGG CCUGUAC C AC CUGCUGGACUGGUUUGC CGUGGAUGAGUC C AAC GAGGUGGACC CCGAGUUCUCUGCCCGGCUGACCGGCAUCAAGCUGGAGAUGGAGCCUUCUCUG AGCUUCUACAACAAGGCCAGAAAUUAUGCCACCAAGAAGCCCUACUCCGUGGA

[0514] GAAGUUCAAGCUGAACUUUCAGCGGCCUACACUGGCCUCUGGCUGGGACGUGA AUAAGGAGAAGAACAAUGGCGCCAUCCUGUUUGUGAAGAACGGCCUGUACUA

[0515] UCUGGGCAUCAUGCCAAAGCAGAAGGGCAGGUAUAAGGCCCUGAGCUUCGAGC

[0516] CCACAGAGAAAACCAGCGAGGGCUUUGAUAAGAUGUACUAUGACUACUUCCCU

[0517] GAUGCCGCCAAGAUGAUCCCAAAGUGCAGCACCCAGCUGAAGGCCGUGACAGC

[0518] CCACUUUCAGACCCACACAACCCCCAUCCUGCUGUCCAACAAUUUCAUCGAGC

[0519] CUCUGGAGAUCACAAAGGAGAUCUACGACCUGAACAAUCCUGAGAAGGAGCCA

[0520] AAGAAGUUUCAGACAGCCUACGCCAAGAAAACCGGCGACCAGAAGGGCUACAG

[0521] AGAGGCCCUGUGCAAGUGGAUCGACUUCACAAGGGAUUUUCUGUCCAAGUAU

[0522] ACCAAGACAACCUCUAUCGAUCUGUCUAGCCUGCGGCCAUCCUCUCAGUAUAA

[0523] GGACCUGGGCGAGUACUAUGCCGAGCUGAAUCCCCUGCUGUACCACAUCAGCU

[0524] UCCAGAGAAUCGCCGAGAAGGAGAUCAUGGAUGCCGUGGAGACAGGCAAGCU

[0525] GUACCUGUUCCAGAUCUAUAACAAGGACUUUGCCAAGGGCCACCACGGCAAGC

[0526] CUAAUCUGCACACACUGUAUUGGACCGGCCUGUUUUCUCCAGAGAACCUGGCC

[0527] AAGACAAGCAUCAAGCUGAAUGGCCAGGCCGAGCUGUUCUACCGCCCUAAGUC

[0528] CAGGAUGAAGAGGAUGGCAGCCCGGCUGGGAGAGAAGAUGCUGAACAAGAAG

[0529] CUGAAGGAUCAGAAAACCCCAAUCCCCGACACCCUGUACCAGGAGCUGUACGA

[0530] CUAUGUGAAUCACAGACUGUCCCACGACCUGUCUGAUGAGGCCAGGGCCCUGC

[0531] UGCCCAACGUGAUCACCAAGGAGGUGUCUCACGAGAUCAUCAAGGAUAGGCGC

[0532] UUUACCAGCGACAAGUUCUUGUUCCACGUGCCUAUCACACUGAACUAUCAGGC

[0533] CGCCAAUUCCCCAUCUAAGUUCAACCAGAGGGUGAAUGCCUACCUGAAGGAGC

[0534] ACCCCGAGACACCUAUCAUCGGCAUCGAUCGGGGCGAGAGAAACCUGAUCUAU

[0535] AUCACAGUGAUCGACUCCACCGGCAAGAUCCUGGAGCAGCGGAGCCUGAACAC

[0536] CAUCCAGCAGUUUGAUUACCAGAAGAAGCUGGACAACAGGGAGAAGGAGAGG

[0537] GUGGCAGCAAGGCAGGCCUGGUCUGUGGUGGGCACAAUCAAGGAUCUGAAGC

[0538] AGGGCUAUCUGAGCCAGGUCAUCCACGAGAUCGUGGACCUGAUGAUCCACUAC

[0539] CAGGCCGUGGUGGUGCUGGAGAACCUGAAUUUCGGCUUUAAGAGCAAGAGGA

[0540] CCGGCAUCGCCGAGAAGGCCGUGUACCAGCAGUUCGAGAAGAUGCUGAUCGAU

[0541] AAGCUGAAUUGCCUGGUGCUGAAGGACUAUCCAGCAGAGAAAGUGGGAGGCG

[0542] UGCUGAACCCAUACCAGCUGACAGACCAGUUCACCUCCUUUGCCAAGAUGGGC

[0543] ACCCAGUCUGGCUUCCUGUUUUACGUGCCUGCCCCAUAUACAUCUAAGAUCGA

[0544] UCCCCUGACCGGCUUCGUGGACCCCUUCGUGUGGAAAACCAUCAAGAAUCACG

[0545] AGAGCCGCAAGCACUUCCUGGAGGGCUUCGACUUUCUGCACUACGACGUGAAA

[0546] ACCGGCGACUUCAUCCUGCACUUUAAGAUGAACAGAAAUCUGUCCUUCCAGAG

[0547] GGGCCUGCCCGGCUUUAUGCCUGCAUGGGAUAUCGUGUUCGAGAAGAACGAG ACACAGUUUGACGCCAAGGGCACCCCUUUCAUCGCCGGCAAGAGAAUCGUGCC

[0548] AGUGAUCGAGAAUCACAGAUUCACCGGCAGAUACCGGGACCUGUAUCCUGCCA

[0549] ACGAGCUGAUCGCCCUGCUGGAGGAGAAGGGCAUCGUGUUCAGGGAUGGCUCC

[0550] AACAUCCUGCCAAAGCUGCUGGAGAAUGACGAUUCUCACGCCAUCGACACCAU

[0551] GGUGGCCCUGAUCCGCAGCGUGCUGCAGAUGCGGAACUCCAAUGCCGCCACAG

[0552] GCGAGGACUAUAUCAACAGCCCCGUGCGCGAUCUGAAUGGCGUGUGCUUCGAC

[0553] UCCCGGUUUCAGAACCCAGAGUGGCCCAUGGACGCCGAUGCCAAUGGCGCCUA

[0554] CCACAUCGCCCUGAAGGGCCAGCUGCUGCUGAAUCACCUGAAGGAGAGCAAGG

[0555] AUCUGAAGCUGCAGAACGGCAUCUCCAAUCAGGACUGGCUGGCCUACAUCCAG

[0556] GAGCUGCGCAACGGUCGUAGCAGUGAUGAUGAAGCAACCGCAGAUAGCCAGCA

[0557] UGCAGCACCGCCCAAGAAGAAGAGGAAAGUCUAAUAGUGAAUGGUUUAUAUU

[0558] GCGGCCGCUUAAUUAAGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCU

[0559] UCUUCUCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUAGG

[0560] AAGUCUAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0561] AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA [SEQ ID NO: 94] mRNA #3

[0562] (5’ Modification = CleanCap® AG 3’0me; Uridine Modifications = N1 -Methyl-

[0563] Pseudo UTP; 3’ Tail Modification = 79A tail)

[0564] AGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCA

[0565] UGACACAGUUCGAGGGCUUUACCAACCUGUAUCAGGUGAGCAAGACACUGCGG

[0566] UUUGAGCUGAUCCCACAGGGCAAGACCCUGAAGCACAUCCAGGAGCAGGGCUU

[0567] CAUCGAGGAGGACAAGGCCCGCAAUGAUCACUACAAGGAGCUGAAGCCCAUCA

[0568] UCGAUCGGAUCUACAAGACCUAUGCCGACCAGUGCCUGCAGCUGGUGCAGCUG

[0569] GAUUGGGAGAACCUGAGCGCCGCCAUCGACUCCUAUAGAAAGGAGAAAACCGA

[0570] GGAGACAAGGAACGCCCUGAUCGAGGAGCAGGCCACAUAUCGCAAUGCCAUCC

[0571] ACGACUACUUCAUCGGCCGGACAGACAACCUGACCGAUGCCAUCAAUAAGAGA

[0572] CACGCCGAGAUCUACAAGGGCCUGUUCAAGGCCGAGCUGUUUAAUGGCAAGGU

[0573] GCUGAAGCAGCUGGGCACCGUGACCACAACCGAGCACGAGAACGCCCUGCUGC

[0574] GGAGCUUCGACAAGUUUACAACCUACUUCUCCGGCUUUUAUGAGAACAGGAA

[0575] GAACGUGUUCAGCGCCGAGGAUAUCAGCACAGCCAUCCCACACCGCAUCGUGC

[0576] AGGACAACUUCCCCAAGUUUAAGGAGAAUUGUCACAUCUUCACACGCCUGAUC

[0577] ACCGCCGUGCCCAGCCUGCGGGAGCACUUUGAGAACGUGAAGAAGGCCAUCGG

[0578] CAUCUUCGUGAGCACCUCCAUCGAGGAGGUGUUUUCCUUCCCUUUUUAUAACC AGCUGCUGACACAGACCCAGAUCGACCUGUAUAACCAGCUGCUGGGAGGAAUC

[0579] UCUCGGGAGGCAGGCACCGAGAAGAUCAAGGGCCUGAACGAGGUGCUGAAUC

[0580] UGGCCAUCCAGAAGAAUGAUGAGACAGCCCACAUCAUCGCCUCCCUGCCACAC

[0581] AGAUUCAUCCCCCUGUUUAAGCAGAUCCUGUCCGAUAGGAACACCCUGUCUUU

[0582] CAUCCUGGAGGAGUUUAAGAGCGACGAGGAAGUGAUCCAGUCCUUCUGCAAG

[0583] UACAAGACACUGCUGAGAAACGAGAACGUGCUGGAGACAGCCGAGGCCCUGUU

[0584] UAACGAGCUGAACAGCAUCGACCUGACACACAUCUUCAUCAGCCACAAGAAGC

[0585] UGGAGACAAUCAGCAGCGCCCUGUGCGACCACUGGGAUACACUGAGGAAUGCC

[0586] CUGUAUGAGCGGAGAAUCUCCGAGCUGACAGGCAAGAUCACCAAGUCUGCCAA

[0587] GGAGAAGGUGCAGCGCAGCCUGAAGCACGAGGAUAUCAACCUGCAGGAGAUC

[0588] AUCUCUGCCGCAGGCAAGGAGCUGAGCGAGGCCUUCAAGCAGAAAACCAGCGA

[0589] GAUCCUGUCCCACGCACACGCCGCCCUGGAUCAGCCACUGCCUACAACCCUGA

[0590] AGAAGCAGGAGGAGAAGGAGAUCCUGAAGUCUCAGCUGGACAGCCUGCUGGG

[0591] CCUGUACCACCUGCUGGACUGGUUUGCCGUGGAUGAGUCCAACGAGGUGGACC

[0592] CCGAGUUCUCUGCCCGGCUGACCGGCAUCAAGCUGGAGAUGGAGCCUUCUCUG

[0593] AGCUUCUACAACAAGGCCAGAAAUUAUGCCACCAAGAAGCCCUACLICCGLIGGA

[0594] GAAGUUCAAGCUGAACUUUCAGCGGCCUACACUGGCCCGUGGCUGGGACGUGA

[0595] AUAAGGAGAAGAACAAUGGCGCCAUCCUGUUUGUGAAGAACGGCCUGUACUA

[0596] UCUGGGCAUCAUGCCAAAGCAGAAGGGCAGGUAUAAGGCCCUGAGCUUCGAGC

[0597] CCACAGAGAAAACCAGCGAGGGCUUUGAUAAGAUGUACUAUGACUACUUCCCU

[0598] GAUGCCGCCAAGAUGAUCCCAAGGUGCAGCACCCAGCUGAAGGCCGUGACAGC

[0599] CCACUUUCAGACCCACACAACCCCCAUCCUGCUGUCCAACAAUUUCAUCGAGC

[0600] CUCUGGAGAUCACAAAGGAGAUCUACGACCUGAACAAUCCUGAGAAGGAGCCA

[0601] AAGAAGUUUCAGACAGCCUACGCCAAGAAAACCGGCGACCAGAAGGGCUACAG

[0602] AGAGGCCCUGUGCAAGUGGAUCGACUUCACAAGGGAUUUUCUGUCCAAGUAU

[0603] ACCAAGACAACCUCUAUCGAUCUGUCUAGCCUGCGGCCAUCCUCUCAGUAUAA

[0604] GGACCUGGGCGAGUACUAUGCCGAGCUGAAUCCCCUGCUGUACCACAUCAGCU

[0605] UCCAGAGAAUCGCCGAGAAGGAGAUCAUGGAUGCCGUGGAGACAGGCAAGCU

[0606] GUACCUGUUCCAGAUCUAUAACAAGGACUUUGCCAAGGGCCACCACGGCAAGC

[0607] CUAAUCUGCACACACUGUAUUGGACCGGCCUGUUUUCUCCAGAGAACCUGGCC

[0608] AAGACAAGCAUCAAGCUGAAUGGCCAGGCCGAGCUGUUCUACCGCCCUAAGUC

[0609] CAGGAUGAAGAGGAUGGCAGCCCGGCUGGGAGAGAAGAUGCUGAACAAGAAG

[0610] CUGAAGGAUCAGAAAACCCCAAUCCCCGACACCCUGUACCAGGAGCUGUACGA

[0611] CUAUGUGAAUCACAGACUGUCCCACGACCUGUCUGAUGAGGCCAGGGCCCUGC UGCCCAACGUGAUCACCAAGGAGGUGUCUCACGAGAUCAUCAAGGAUAGGCGC

[0612] UUUACCAGCGACAAGUUCUUGUUCCACGUGCCUAUCACACUGAACUAUCAGGC

[0613] CGCCAAUUCCCCAUCUAAGUUCAACCAGAGGGUGAAUGCCUACCUGAAGGAGC

[0614] ACCCCGAGACACCUAUCAUCGGCAUCGAUCGGGGCGAGAGAAACCUGAUCUAU

[0615] AUCACAGUGAUCGACUCCACCGGCAAGAUCCUGGAGCAGCGGAGCCUGAACAC

[0616] CAUCCAGCAGUUUGAUUACCAGAAGAAGCUGGACAACAGGGAGAAGGAGAGG

[0617] GUGGCAGCAAGGCAGGCCUGGUCUGUGGUGGGCACAAUCAAGGAUCUGAAGC

[0618] AGGGCUAUCUGAGCCAGGUCAUCCACGAGAUCGUGGACCUGAUGAUCCACUAC

[0619] CAGGCCGUGGUGGUGCUGGAGAACCUGAAUUUCGGCUUUAAGAGCAAGAGGA

[0620] CCGGCAUCGCCGAGAAGGCCGUGUACCAGCAGUUCGAGAAGAUGCUGAUCGAU

[0621] AAGCUGAAUUGCCUGGUGCUGAAGGACUAUCCAGCAGAGAAAGUGGGAGGCG

[0622] UGCUGAACCCAUACCAGCUGACAGACCAGUUCACCUCCUUUGCCAAGAUGGGC

[0623] ACCCAGUCUGGCUUCCUGUUUUACGUGCCUGCCCCAUAUACAUCUAAGAUCGA

[0624] UCCCCUGACCGGCUUCGUGGACCCCUUCGUGUGGAAAACCAUCAAGAAUCACG

[0625] AGAGCCGCAAGCACUUCCUGGAGGGCUUCGACUUUCUGCACUACGACGUGAAA

[0626] ACCGGCGACUUCAUCCUGCACUUUAAGAUGAACAGAAAUCUGUCCUUCCAGAG

[0627] GGGCCUGCCCGGCUUUAUGCCUGCAUGGGAUAUCGUGUUCGAGAAGAACGAG

[0628] ACACAGUUUGACGCCAAGGGCACCCCUUUCAUCGCCGGCAAGAGAAUCGUGCC

[0629] AGUGAUCGAGAAUCACAGAUUCACCGGCAGAUACCGGGACCUGUAUCCUGCCA

[0630] ACGAGCUGAUCGCCCUGCUGGAGGAGAAGGGCAUCGUGUUCAGGGAUGGCUCC

[0631] AACAUCCUGCCAAAGCUGCUGGAGAAUGACGAUUCUCACGCCAUCGACACCAU

[0632] GGUGGCCCUGAUCCGCAGCGUGCUGCAGAUGCGGAACUCCAAUGCCGCCACAG

[0633] GCGAGGACUAUAUCAACAGCCCCGUGCGCGAUCUGAAUGGCGUGUGCUUCGAC

[0634] UCCCGGUUUCAGAACCCAGAGUGGCCCAUGGACGCCGAUGCCAAUGGCGCCUA

[0635] CCACAUCGCCCUGAAGGGCCAGCUGCUGCUGAAUCACCUGAAGGAGAGCAAGG

[0636] AUCUGAAGCUGCAGAACGGCAUCUCCAAUCAGGACUGGCUGGCCUACAUCCAG

[0637] GAGCUGCGCAACGGCGGAUCCCCUGCUGCUAAACGUGUUAAGCUUGAUGGGGG

[0638] UAGCCCGGCAGCCAAGAGAGUCAAACUCGACUAGAUGGUUUAUAUUGCGGCCG

[0639] CUUAAUUAAGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUC

[0640] UCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUAGGAAGUCUA

[0641] GAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0642] AAAAAAAAAAAAAAAAAAAAAAAAAAAA [SEQ ID NO: 95] mRNA #4 (5’ Modification = CleanCap® AG 3’0me; Uridine Modifications = Nl-Methyl- Pseudo UTP; 3’ Tail Modification = 79A tail)

[0643] AGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCA

[0644] UGACACAGUUCGAGGGCUUUACCAACCUGUAUCAGGUGAGCAAGACACUGCGG UUUGAGCUGAUCCCACAGGGCAAGACCCUGAAGCACAUCCAGGAGCAGGGCUU CAUCGAGGAGGACAAGGCCCGCAAUGAUCACUACAAGGAGCUGAAGCCCAUCA UCGAUCGGAUCUACAAGACCUAUGCCGACCAGUGCCUGCAGCUGGUGCAGCUG GAUUGGGAGAACCUGAGCGCCGCCAUCGACUCCUAUAGAAAGGAGAAAACCGA GGAGACAAGGAACGCCCUGAUCGAGGAGCAGGCCACAUAUCGCAAUGCCAUCC ACGACUACUUCAUCGGCCGGACAGACAACCUGACCGAUGCCAUCAAUAAGAGA CACGCCGAGAUCUACAAGGGCCUGUUCAAGGCCGAGCUGUUUAAUGGCAAGGU GCUGAAGCAGCUGGGCACCGUGACCACAACCGAGCACGAGAACGCCCUGCUGC GGAGCUUCGACAAGUUUACAACCUACUUCUCCGGCUUUUAUGAGAACAGGAA GAACGUGUUCAGCGCCGAGGAUAUCAGCACAGCCAUCCCACACCGCAUCGUGC AGGACAACUUCCCCAAGUUUAAGGAGAAUUGUCACAUCUUCACACGCCUGAUC ACCGCCGUGCCCAGCCUGCGGGAGCACUUUGAGAACGUGAAGAAGGCCAUCGG CAUCUUCGUGAGCACCUCCAUCGAGGAGGUGUUUUCCUUCCCUUUUUAUAACC AGCUGCUGACACAGACCCAGAUCGACCUGUAUAACCAGCUGCUGGGAGGAAUC UCUCGGGAGGCAGGCACCGAGAAGAUCAAGGGCCUGAACGAGGUGCUGAAUC UGGCCAUCCAGAAGAAUGAUGAGACAGCCCACAUCAUCGCCUCCCUGCCACAC AGAUUCAUCCCCCUGUUUAAGCAGAUCCUGUCCGAUAGGAACACCCUGUCUUU CAUCCUGGAGGAGUUUAAGAGCGACGAGGAAGUGAUCCAGUCCUUCUGCAAG UACAAGACACUGCUGAGAAACGAGAACGUGCUGGAGACAGCCGAGGCCCUGUU UAACGAGCUGAACAGCAUCGACCUGACACACAUCUUCAUCAGCCACAAGAAGC UGGAGACAAUCAGCAGCGCCCUGUGCGACCACUGGGAUACACUGAGGAAUGCC

[0645] CUGUAUGAGCGGAGAAUCUCCGAGCUGACAGGCAAGAUCACCAAGUCUGCCAA GGAGAAGGUGCAGCGCAGCCUGAAGCACGAGGAUAUCAACCUGCAGGAGAUC AUCUCUGCCGCAGGCAAGGAGCUGAGCGAGGCCUUCAAGCAGAAAACCAGCGA GAUCCUGUCCCACGCACACGCCGCCCUGGAUCAGCCACUGCCUACAACCCUGA AGAAGCAGGAGGAGAAGGAGAUCCUGAAGUCUCAGCUGGACAGCCUGCUGGG CCUGUAC C AC CUGCUGGACUGGUUUGC CGUGGAUGAGUC C AAC GAGGUGGACC CCGAGUUCUCUGCCCGGCUGACCGGCAUCAAGCUGGAGAUGGAGCCUUCUCUG AGCUUCUACAACAAGGCCAGAAAUUAUGCCACCAAGAAGCCCUACUCCGUGGA

[0646] GAAGUUCAAGCUGAACUUUCAGCGGCCUACACUGGCCCGUGGCUGGGACGUGA AUGUGGAGAAGAACCGUGGCGCCAUCCUGUUUGUGAAGAACGGCCUGUACUA

[0647] UCUGGGCAUCAUGCCAAAGCAGAAGGGCAGGUAUAAGGCCCUGAGCUUCGAGC

[0648] CCACAGAGAAAACCAGCGAGGGCUUUGAUAAGAUGUACUAUGACUACUUCCCU

[0649] GAUGCCGCCAAGAUGAUCCCAAAGUGCAGCACCCAGCUGAAGGCCGUGACAGC

[0650] CCACUUUCAGACCCACACAACCCCCAUCCUGCUGUCCAACAAUUUCAUCGAGC

[0651] CUCUGGAGAUCACAAAGGAGAUCUACGACCUGAACAAUCCUGAGAAGGAGCCA

[0652] AAGAAGUUUCAGACAGCCUACGCCAAGAAAACCGGCGACCAGAAGGGCUACAG

[0653] AGAGGCCCUGUGCAAGUGGAUCGACUUCACAAGGGAUUUUCUGUCCAAGUAU

[0654] ACCAAGACAACCUCUAUCGAUCUGUCUAGCCUGCGGCCAUCCUCUCAGUAUAA

[0655] GGACCUGGGCGAGUACUAUGCCGAGCUGAAUCCCCUGCUGUACCACAUCAGCU

[0656] UCCAGAGAAUCGCCGAGAAGGAGAUCAUGGAUGCCGUGGAGACAGGCAAGCU

[0657] GUACCUGUUCCAGAUCUAUAACAAGGACUUUGCCAAGGGCCACCACGGCAAGC

[0658] CUAAUCUGCACACACUGUAUUGGACCGGCCUGUUUUCUCCAGAGAACCUGGCC

[0659] AAGACAAGCAUCAAGCUGAAUGGCCAGGCCGAGCUGUUCUACCGCCCUAAGUC

[0660] CAGGAUGAAGAGGAUGGCAGCCCGGCUGGGAGAGAAGAUGCUGAACAAGAAG

[0661] CUGAAGGAUCAGAAAACCCCAAUCCCCGACACCCUGUACCAGGAGCUGUACGA

[0662] CUAUGUGAAUCACAGACUGUCCCACGACCUGUCUGAUGAGGCCAGGGCCCUGC

[0663] UGCCCAACGUGAUCACCAAGGAGGUGUCUCACGAGAUCAUCAAGGAUAGGCGC

[0664] UUUACCAGCGACAAGUUCUUGUUCCACGUGCCUAUCACACUGAACUAUCAGGC

[0665] CGCCAAUUCCCCAUCUAAGUUCAACCAGAGGGUGAAUGCCUACCUGAAGGAGC

[0666] ACCCCGAGACACCUAUCAUCGGCAUCGAUCGGGGCGAGAGAAACCUGAUCUAU

[0667] AUCACAGUGAUCGACUCCACCGGCAAGAUCCUGGAGCAGCGGAGCCUGAACAC

[0668] CAUCCAGCAGUUUGAUUACCAGAAGAAGCUGGACAACAGGGAGAAGGAGAGG

[0669] GUGGCAGCAAGGCAGGCCUGGUCUGUGGUGGGCACAAUCAAGGAUCUGAAGC

[0670] AGGGCUAUCUGAGCCAGGUCAUCCACGAGAUCGUGGACCUGAUGAUCCACUAC

[0671] CAGGCCGUGGUGGUGCUGGAGAACCUGAAUUUCGGCUUUAAGAGCAAGAGGA

[0672] CCGGCAUCGCCGAGAAGGCCGUGUACCAGCAGUUCGAGAAGAUGCUGAUCGAU

[0673] AAGCUGAAUUGCCUGGUGCUGAAGGACUAUCCAGCAGAGAAAGUGGGAGGCG

[0674] UGCUGAACCCAUACCAGCUGACAGACCAGUUCACCUCCUUUGCCAAGAUGGGC

[0675] ACCCAGUCUGGCUUCCUGUUUUACGUGCCUGCCCCAUAUACAUCUAAGAUCGA

[0676] UCCCCUGACCGGCUUCGUGGACCCCUUCGUGUGGAAAACCAUCAAGAAUCACG

[0677] AGAGCCGCAAGCACUUCCUGGAGGGCUUCGACUUUCUGCACUACGACGUGAAA

[0678] ACCGGCGACUUCAUCCUGCACUUUAAGAUGAACAGAAAUCUGUCCUUCCAGAG

[0679] GGGCCUGCCCGGCUUUAUGCCUGCAUGGGAUAUCGUGUUCGAGAAGAACGAG ACACAGUUUGACGCCAAGGGCACCCCUUUCAUCGCCGGCAAGAGAAUCGUGCC AGUGAUCGAGAAUCACAGAUUCACCGGCAGAUACCGGGACCUGUAUCCUGCCA

[0680] ACGAGCUGAUCGCCCUGCUGGAGGAGAAGGGCAUCGUGUUCAGGGAUGGCUCC AACAUCCUGCCAAAGCUGCUGGAGAAUGACGAUUCUCACGCCAUCGACACCAU

[0681] GGUGGCCCUGAUCCGCAGCGUGCUGCAGAUGCGGAACUCCAAUGCCGCCACAG GCGAGGACUAUAUCAACAGCCCCGUGCGCGAUCUGAAUGGCGUGUGCUUCGAC

[0682] UCCCGGUUUCAGAACCCAGAGUGGCCCAUGGACGCCGAUGCCAAUGGCGCCUA CCACAUCGCCCUGAAGGGCCAGCUGCUGCUGAAUCACCUGAAGGAGAGCAAGG AUCUGAAGCUGCAGAACGGCAUCUCCAAUCAGGACUGGCUGGCCUACAUCCAG

[0683] GAGCUGCGCAACGGCGGAUCCCCUGCUGCUAAACGUGUUAAGCUUGAUGGGGG

[0684] UAGCCCGGCAGCCAAGAGAGUCAAACUCGACUAGAUGGUUUAUAUUGCGGCCG CUUAAUUAAGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUC UCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUAGGAAGUCUA

[0685] GAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0686] AAAAAAAAAAAAAAAAAAAAAAAAAAAA [SEQ ID NO: 96] mRNA #5

[0687] (5’ Modification = CleanCap® AG 3’0me; Uridine Modifications = N1 -Methyl- Pseudo UTP; 3’ Tail Modification = 79A tail)

[0688] AGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCA

[0689] UGACACAGUUCGAGGGCUUUACCAACCUGUAUCAGGUGAGCAAGACACUGCGG UUUGAGCUGAUCCCACAGGGCAAGACCCUGAAGCACAUCCAGGAGCAGGGCUU CAUCGAGGAGGACAAGGCCCGCAAUGAUCACUACAAGGAGCUGAAGCCCAUCA UCGAUCGGAUCUACAAGACCUAUGCCGACCAGUGCCUGCAGCUGGUGCAGCUG GAUUGGGAGAACCUGAGCGCCGCCAUCGACUCCUAUAGAAAGGAGAAAACCGA GGAGACAAGGAACGCCCUGAUCGAGGAGCAGGCCACAUAUCGCAAUGCCAUCC ACGACUACUUCAUCGGCCGGACAGACAACCUGACCGAUGCCAUCAAUAAGAGA CACGCCGAGAUCUACAAGGGCCUGUUCAAGGCCGAGCUGUUUAAUGGCAAGGU GCUGAAGCAGCUGGGCACCGUGACCACAACCGAGCACGAGAACGCCCUGCUGC GGAGCUUCGACAAGUUUACAACCUACUUCUCCGGCUUUUAUGAAAACAGGAA GAACGUGUUCAGCGCCGAGGAUAUCAGCACAGCCAUCCCACACCGCAUCGUGC AGGACAACUUCCCCAAGUUUAAGGAGAAUUGUCACAUCUUCACACGCCUGAUC ACCGCCGUGCCCAGCCUGCGGGAGCACUUUGAGAACGUGAAGAAGGCCAUCGG

[0690] CAUCUUCGUGAGCACCUCCAUCGAGGAGGUGUUUUCCUUCCCUUUUUAUAACC AGCUGCUGACACAGACCCAGAUCGACCUGUAUAACCAGCUGCUGGGAGGAAUC

[0691] UCUCGGGAGGCAGGCACCGAGAAGAUCAAGGGCCUGAACGAGGUGCUGAAUC

[0692] UGGCCAUCCAGAAGAAUGAUGAGACAGCCCACAUCAUCGCCUCCCUGCCACAC

[0693] AGAUUCAUCCCCCUGUUUAAGCAGAUCCUGUCCGAUAGGAACACCCUGUCUUU

[0694] CAUCCUGGAGGAGUUUAAGAGCGACGAGGAAGUGAUCCAGUCCUUCUGCAAG

[0695] UACAAGACACUGCUGAGAAACGAGAACGUGCUGGAGACAGCCGAGGCCCUGUU

[0696] UAACGAGCUGAACAGCAUCGACCUGACACACAUCUUCAUCAGCCACAAGAAGC

[0697] UGGAGACAAUCAGCAGCGCCCUGUGCGACCACUGGGAUACACUGAGGAAUGCC

[0698] CUGUAUGAGCGGAGAAUCUCCGAGCUGACAGGCAAGAUCACCAAGUCUGCCAA

[0699] GGAGAAGGUGCAGCGCAGCCUGAAGCACGAGGAUAUCAACCUGCAGGAGAUC

[0700] AUCUCUGCCGCAGGCAAGGAGCUGAGCGAGGCCUUCAAGCAGAAAACCAGCGA

[0701] GAUCCUGUCCCACGCACACGCCGCCCUGGAUCAGCCACUGCCUACAACCCUGA

[0702] AGAAGCAGGAGGAGAAGGAGAUCCUGAAGUCUCAGCUGGACAGCCUGCUGGG

[0703] CCUGUACCACCUGCUGGACUGGUUUGCCGUGGAUGAGUCCAACGAGGUGGACC

[0704] CCGAGUUCUCUGCCCGGCUGACCGGCAUCAAGCUGGAGAUGGAGCCUUCUCUG

[0705] AGCUUCUACAACAAGGCCAGAAAUUAUGCCACCAAGAAGCCCUACLICCGLIGGA

[0706] GAAGUUCAAGCUGAACUUUCAGCGGCCUACACUGGCCUCUGGCUGGGACGUGA

[0707] AUAAGGAGAAGAACAAUGGCGCCAUCCUGUUUGUGAAGAACGGCCUGUACUA

[0708] UCUGGGCAUCAUGCCAAAGCAGAAGGGCAGGUAUAAGGCCCUGAGCUUCGAGC

[0709] CCACAGAGAAAACCAGCGAGGGCUUUGAUAAGAUGUACUAUGACUACUUCCCU

[0710] GAUGCCGCCAAGAUGAUCCCAAAGUGCAGCACCCAGCUGAAGGCCGUGACAGC

[0711] CCACUUUCAGACCCACACAACCCCCAUCCUGCUGUCCAACAAUUUCAUCGAGC

[0712] CUCUGGAGAUCACAAAGGAGAUCUACGACCUGAACAAUCCUGAGAAGGAGCCA

[0713] AAGAAGUUUCAGACAGCCUACGCCAAGAAAACCGGCGACCAGAAGGGCUACAG

[0714] AGAGGCCCUGUGCAAGUGGAUCGACUUCACAAGGGAUUUUCUGUCCAAGUAU

[0715] ACCAAGACAACCUCUAUCGAUCUGUCUAGCCUGCGGCCAUCCUCUCAGUAUAA

[0716] GGACCUGGGCGAGUACUAUGCCGAGCUGAAUCCCCUGCUGUACCACAUCAGCU

[0717] UCCAGAGAAUCGCCGAGAAGGAGAUCAUGGAUGCCGUGGAGACAGGCAAGCU

[0718] GUACCUGUUCCAGAUCUAUAACAAGGACUUUGCCAAGGGCCACCACGGCAAGC

[0719] CUAAUCUGCACACACUGUAUUGGACCGGCCUGUUUUCUCCAGAGAACCUGGCC

[0720] AAGACAAGCAUCAAGCUGAAUGGCCAGGCCGAGCUGUUCUACCGCCCUAAGUC

[0721] CAGGAUGAAGAGGAUGGCAGCUCGGCUGGGAGAGAAGAUGCUGAACAAGAAG

[0722] CUGAAGGAUCAGAAAACCCCAAUCCCCGACACCCUGUACCAGGAGCUGUACGA

[0723] CUAUGUGAAUCACAGACUGUCCCACGACCUGUCUGAUGAGGCCAGGGCCCUGC UGCCCAACGUGAUCACCAAGGAGGUGUCUCACGAGAUCAUCAAGGAUAGGCGC

[0724] UUUACCAGCGACAAGUUCUUGUUCCACGUGCCUAUCACACUGAACUAUCAGGC

[0725] CGCCAAUUCCCCAUCUAAGUUCAACCAGAGGGUGAAUGCCUACCUGAAGGAGC

[0726] ACCCCGAGACACCUAUCAUCGGCAUCGAUCGGGGCGAGAGAAACCUGAUCUAU

[0727] AUCACAGUGAUCGACUCCACCGGCAAGAUCCUGGAGCAGCGGAGCCUGAACAC

[0728] CAUCCAGCAGUUUGAUUACCAGAAGAAGCUGGACAACAGGGAGAAGGAGAGG

[0729] GUGGCAGCAAGGCAGGCCUGGUCUGUGGUGGGCACAAUCAAGGAUCUGAAGC

[0730] AGGGCUAUCUGAGCCAGGUCAUCCACGAGAUCGUGGACCUGAUGAUCCACUAC

[0731] CAGGCCGUGGUGGUGCUGGAGAACCUGAAUUUCGGCUUUAAGAGCAAGAGGA

[0732] CCGGCAUCGCCGAGAAGGCCGUGUACCAGCAGUUCGAGAAGAUGCUGAUCGAU

[0733] AAGCUGAAUUGCCUGGUGCUGAAGGACUAUCCAGCAGAGAAAGUGGGAGGCG

[0734] UGCUGAACCCAUACCAGCUGACAGACCAGUUCACCUCCUUUGCCAAGAUGGGC

[0735] ACCCAGUCUGGCUUCCUGUUUUACGUGCCUGCCCCAUAUACAUCUAAGAUCGA

[0736] UCCCCUGACCGGCUUCGUGGACCCCUUCGUGUGGAAAACCAUCAAGAAUCACG

[0737] AGAGCCGCAAGCACUUCCUGGAGGGCUUCGACUUUCUGCACUACGACGUGAAA

[0738] ACCGGCGACUUCAUCCUGCACUUUAAGAUGAACAGAAAUCUGUCCUUCCAGAG

[0739] GGGCCUGCCCGGCUUUAUGCCUGCAUGGGAUAUCGUGUUCGAGAAGAACGAG

[0740] ACACAGUUUGACGCCAAGGGCACCCCUUUCAUCGCCGGCAAGAGAAUCGUGCC

[0741] AGUGAUCGAGAAUCACAGAUUCACCGGCAGAUACCGGGACCUGUAUCCUGCCA

[0742] ACGAGCUGAUCGCCCUGCUGGAGGAGAAGGGCAUCGUGUUCAGGGAUGGCUCC

[0743] AACAUCCUGCCAAAGCUGCUGGAGAAUGACGAUUCUCACGCCAUCGACACCAU

[0744] GGUGGCCCUGAUCCGCAGCGUGCUGCAGAUGCGGAACUCCAAUGCCGCCACAG

[0745] GCGAGGACUAUAUCAACAGCCCCGUGCGCGAUCUGAAUGGCGUGUGCUUCGAC

[0746] UCCCGGUUUCAGAACCCAGAGUGGCCCAUGGACGCCGAUGCCAAUGGCGCCUA

[0747] CCACAUCGCCCUGAAGGGCCAGCUGCUGCUGAAUCACCUGAAGGAGAGCAAGG

[0748] AUCUGAAGCUGCAGAACGGCAUCUCCAAUCAGGACUGGCUGGCCUACAUCCAG

[0749] GAGCUGCGCAACGGCGGAUCCCCUGCUGCUAAACGUGUUAAGCUUGAUGGGGG

[0750] UAGCCCGGCAGCCAAGAGAGUCAAACUCGACUAGAUGGUUUAUAUUGCGGCCG

[0751] CUUAAUUAAGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUC

[0752] UCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUAGGAAGUCUA

[0753] GAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0754] AAAAAAAAAAAAAAAAAAAAAAAAAAAA [SEQ ID NO: 97] mRNA #6 (Uridine Modifications = N1 -Methyl-Pseudo UTP; 3’ Tail Modification = 79A tail)

[0755] AGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCA

[0756] UGACACAGUUCGAGGGCUUUACCAACCUGUAUCAGGUGAGCAAGACACUGCGG

[0757] UUUGAGCUGAUCCCACAGGGCAAGACCCUGAAGCACAUCCAGGAGCAGGGCUU

[0758] CAUCGAGGAGGACAAGGCCCGCAAUGAUCACUACAAGGAGCUGAAGCCCAUCA

[0759] UCGAUCGGAUCUACAAGACCUAUGCCGACCAGUGCCUGCAGCUGGUGCAGCUG

[0760] GAUUGGGAGAACCUGAGCGCCGCCAUCGACUCCUAUAGAAAGGAGAAAACCGA

[0761] GGAGACAAGGAACGCCCUGAUCGAGGAGCAGGCCACAUAUCGCAAUGCCAUCC

[0762] ACGACUACUUCAUCGGCCGGACAGACAACCUGACCGAUGCCAUCAAUAAGAGA

[0763] CACGCCGAGAUCUACAAGGGCCUGUUCAAGGCCGAGCUGUUUAAUGGCAAGGU

[0764] GCUGAAGCAGCUGGGCACCGUGACCACAACCGAGCACGAGAACGCCCUGCUGC

[0765] GGAGCUUCGACAAGUUUACAACCUACUUCUCCGGCUUUUAUGAAAACAGGAA

[0766] GAACGUGUUCAGCGCCGAGGAUAUCAGCACAGCCAUCCCACACCGCAUCGUGC

[0767] AGGACAACUUCCCCAAGUUUAAGGAGAAUUGUCACAUCUUCACACGCCUGAUC

[0768] ACCGCCGUGCCCAGCCUGCGGGAGCACUUUGAGAACGUGAAGAAGGCCAUCGG

[0769] CAUCUUCGUGAGCACCUCCAUCGAGGAGGUGUUUUCCUUCCCUUUUUAUAACC

[0770] AGCUGCUGACACAGACCCAGAUCGACCUGUAUAACCAGCUGCUGGGAGGAAUC

[0771] UCUCGGGAGGCAGGCACCGAGAAGAUCAAGGGCCUGAACGAGGUGCUGAAUC

[0772] UGGCCAUCCAGAAGAAUGAUGAGACAGCCCACAUCAUCGCCUCCCUGCCACAC

[0773] AGAUUCAUCCCCCUGUUUAAGCAGAUCCUGUCCGAUAGGAACACCCUGUCUUU

[0774] CAUCCUGGAGGAGUUUAAGAGCGACGAGGAAGUGAUCCAGUCCUUCUGCAAG

[0775] UACAAGACACUGCUGAGAAACGAGAACGUGCUGGAGACAGCCGAGGCCCUGUU

[0776] UAACGAGCUGAACAGCAUCGACCUGACACACAUCUUCAUCAGCCACAAGAAGC

[0777] UGGAGACAAUCAGCAGCGCCCUGUGCGACCACUGGGAUACACUGAGGAAUGCC

[0778] CUGUAUGAGCGGAGAAUCUCCGAGCUGACAGGCAAGAUCACCAAGUCUGCCAA

[0779] GGAGAAGGUGCAGCGCAGCCUGAAGCACGAGGAUAUCAACCUGCAGGAGAUC

[0780] AUCUCUGCCGCAGGCAAGGAGCUGAGCGAGGCCUUCAAGCAGAAAACCAGCGA

[0781] GAUCCUGUCCCACGCACACGCCGCCCUGGAUCAGCCACUGCCUACAACCCUGA

[0782] AGAAGCAGGAGGAGAAGGAGAUCCUGAAGUCUCAGCUGGACAGCCUGCUGGG

[0783] CCUGUACCACCUGCUGGACUGGUUUGCCGUGGAUGAGUCCAACGAGGUGGACC

[0784] CCGAGUUCUCUGCCCGGCUGACCGGCAUCAAGCUGGAGAUGGAGCCUUCUCUG

[0785] AGCUUCUACAACAAGGCCAGAAAUUAUGCCACCAAGAAGCCCUACUCCGUGGA

[0786] GAAGUUCAAGCUGAACUUUCAGCGGCCUACACUGGCCUCUGGCUGGGACGUGA

[0787] AUAAGGAGAAGAACAAUGGCGCCAUCCUGUUUGUGAAGAACGGCCUGUACUA UCUGGGCAUCAUGCCAAAGCAGAAGGGCAGGUAUAAGGCCCUGAGCUUCGAGC

[0788] CCACAGAGAAAACCAGCGAGGGCUUUGAUAAGAUGUACUAUGACUACUUCCCU

[0789] GAUGCCGCCAAGAUGAUCCCAAAGUGCAGCACCCAGCUGAAGGCCGUGACAGC

[0790] CCACUUUCAGACCCACACAACCCCCAUCCUGCUGUCCAACAAUUUCAUCGAGC

[0791] CUCUGGAGAUCACAAAGGAGAUCUACGACCUGAACAAUCCUGAGAAGGAGCCA

[0792] AAGAAGUUUCAGACAGCCUACGCCAAGAAAACCGGCGACCAGAAGGGCUACAG

[0793] AGAGGCCCUGUGCAAGUGGAUCGACUUCACAAGGGAUUUUCUGUCCAAGUAU

[0794] ACCAAGACAACCUCUAUCGAUCUGUCUAGCCUGCGGCCAUCCUCUCAGUAUAA

[0795] GGACCUGGGCGAGUACUAUGCCGAGCUGAAUCCCCUGCUGUACCACAUCAGCU

[0796] UCCAGAGAAUCGCCGAGAAGGAGAUCAUGGAUGCCGUGGAGACAGGCAAGCU

[0797] GUACCUGUUCCAGAUCUAUAACAAGGACUUUGCCAAGGGCCACCACGGCAAGC

[0798] CUAAUCUGCACACACUGUAUUGGACCGGCCUGUUUUCUCCAGAGAACCUGGCC

[0799] AAGACAAGCAUCAAGCUGAAUGGCCAGGCCGAGCUGUUCUACCGCCCUAAGUC

[0800] CAGGAUGAAGAGGAUGGCAGCUCGGCUGGGAGAGAAGAUGCUGAACAAGAAG

[0801] CUGAAGGAUCAGAAAACCCCAAUCCCCGACACCCUGUACCAGGAGCUGUACGA

[0802] CUAUGUGAAUCACAGACUGUCCCACGACCUGUCUGAUGAGGCCAGGGCCCUGC

[0803] UGCCCAACGUGAUCACCAAGGAGGUGUCUCACGAGAUCAUCAAGGAUAGGCGC

[0804] UUUACCAGCGACAAGUUCUUGUUCCACGUGCCUAUCACACUGAACUAUCAGGC

[0805] CGCCAAUUCCCCAUCUAAGUUCAACCAGAGGGUGAAUGCCUACCUGAAGGAGC

[0806] ACCCCGAGACACCUAUCAUCGGCAUCGAUCGGGGCGAGAGAAACCUGAUCUAU

[0807] AUCACAGUGAUCGACUCCACCGGCAAGAUCCUGGAGCAGCGGAGCCUGAACAC

[0808] CAUCCAGCAGUUUGAUUACCAGAAGAAGCUGGACAACAGGGAGAAGGAGAGG

[0809] GUGGCAGCAAGGCAGGCCUGGUCUGUGGUGGGCACAAUCAAGGAUCUGAAGC

[0810] AGGGCUAUCUGAGCCAGGUCAUCCACGAGAUCGUGGACCUGAUGAUCCACUAC

[0811] CAGGCCGUGGUGGUGCUGGAGAACCUGAAUUUCGGCUUUAAGAGCAAGAGGA

[0812] CCGGCAUCGCCGAGAAGGCCGUGUACCAGCAGUUCGAGAAGAUGCUGAUCGAU

[0813] AAGCUGAAUUGCCUGGUGCUGAAGGACUAUCCAGCAGAGAAAGUGGGAGGCG

[0814] UGCUGAACCCAUACCAGCUGACAGACCAGUUCACCUCCUUUGCCAAGAUGGGC

[0815] ACCCAGUCUGGCUUCCUGUUUUACGUGCCUGCCCCAUAUACAUCUAAGAUCGA

[0816] UCCCCUGACCGGCUUCGUGGACCCCUUCGUGUGGAAAACCAUCAAGAAUCACG

[0817] AGAGCCGCAAGCACUUCCUGGAGGGCUUCGACUUUCUGCACUACGACGUGAAA

[0818] ACCGGCGACUUCAUCCUGCACUUUAAGAUGAACAGAAAUCUGUCCUUCCAGAG

[0819] GGGCCUGCCCGGCUUUAUGCCUGCAUGGGAUAUCGUGUUCGAGAAGAACGAG

[0820] ACACAGUUUGACGCCAAGGGCACCCCUUUCAUCGCCGGCAAGAGAAUCGUGCC AGUGAUCGAGAAUCACAGAUUCACCGGCAGAUACCGGGACCUGUAUCCUGCCA

[0821] ACGAGCUGAUCGCCCUGCUGGAGGAGAAGGGCAUCGUGUUCAGGGAUGGCUCC

[0822] AACAUCCUGCCAAAGCUGCUGGAGAAUGACGAUUCUCACGCCAUCGACACCAU

[0823] GGUGGCCCUGAUCCGCAGCGUGCUGCAGAUGCGGAACUCCAAUGCCGCCACAG

[0824] GCGAGGACUAUAUCAACAGCCCCGUGCGCGAUCUGAAUGGCGUGUGCUUCGAC

[0825] UCCCGGUUUCAGAACCCAGAGUGGCCCAUGGACGCCGAUGCCAAUGGCGCCUA

[0826] CCACAUCGCCCUGAAGGGCCAGCUGCUGCUGAAUCACCUGAAGGAGAGCAAGG

[0827] AUCUGAAGCUGCAGAACGGCAUCUCCAAUCAGGACUGGCUGGCCUACAUCCAG

[0828] GAGCUGCGCAACGGCGGAUCCCCUGCUGCUAAACGUGUUAAGCUUGAUGGGGG

[0829] UAGCCCGGCAGCCAAGAGAGUCAAACUCGACUAGAUGGUUUAUAUUGCGGCCG

[0830] CUUAAUUAAGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUC

[0831] UCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUAGGAAGUCUA

[0832] GAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0833] AAAAAAAAAAAAAAAAAAAAAAAAAAAA [SEQ ID NO: 433] mRNA #7

[0834] (5’ Modification = CleanCap® AG 3’0me; Uridine Modifications = Nl-Methyl- Pseudo UTP)

[0835] AGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCA

[0836] UGACACAGUUCGAGGGCUUUACCAACCUGUAUCAGGUGAGCAAGACACUGCGG

[0837] UUUGAGCUGAUCCCACAGGGCAAGACCCUGAAGCACAUCCAGGAGCAGGGCUU

[0838] CAUCGAGGAGGACAAGGCCCGCAAUGAUCACUACAAGGAGCUGAAGCCCAUCA

[0839] UCGAUCGGAUCUACAAGACCUAUGCCGACCAGUGCCUGCAGCUGGUGCAGCUG

[0840] GAUUGGGAGAACCUGAGCGCCGCCAUCGACUCCUAUAGAAAGGAGAAAACCGA

[0841] GGAGACAAGGAACGCCCUGAUCGAGGAGCAGGCCACAUAUCGCAAUGCCAUCC

[0842] ACGACUACUUCAUCGGCCGGACAGACAACCUGACCGAUGCCAUCAAUAAGAGA

[0843] CACGCCGAGAUCUACAAGGGCCUGUUCAAGGCCGAGCUGUUUAAUGGCAAGGU

[0844] GCUGAAGCAGCUGGGCACCGUGACCACAACCGAGCACGAGAACGCCCUGCUGC

[0845] GGAGCUUCGACAAGUUUACAACCUACUUCUCCGGCUUUUAUGAAAACAGGAA

[0846] GAACGUGUUCAGCGCCGAGGAUAUCAGCACAGCCAUCCCACACCGCAUCGUGC

[0847] AGGACAACUUCCCCAAGUUUAAGGAGAAUUGUCACAUCUUCACACGCCUGAUC

[0848] ACCGCCGUGCCCAGCCUGCGGGAGCACUUUGAGAACGUGAAGAAGGCCAUCGG

[0849] CAUCUUCGUGAGCACCUCCAUCGAGGAGGUGUUUUCCUUCCCUUUUUAUAACC

[0850] AGCUGCUGACACAGACCCAGAUCGACCUGUAUAACCAGCUGCUGGGAGGAAUC UCUCGGGAGGCAGGCACCGAGAAGAUCAAGGGCCUGAACGAGGUGCUGAAUC UGGCCAUCCAGAAGAAUGAUGAGACAGCCCACAUCAUCGCCUCCCUGCCACAC AGAUUCAUCCCCCUGUUUAAGCAGAUCCUGUCCGAUAGGAACACCCUGUCUUU CAUCCUGGAGGAGUUUAAGAGCGACGAGGAAGUGAUCCAGUCCUUCUGCAAG UACAAGACACUGCUGAGAAACGAGAACGUGCUGGAGACAGCCGAGGCCCUGUU UAACGAGCUGAACAGCAUCGACCUGACACACAUCUUCAUCAGCCACAAGAAGC UGGAGACAAUCAGCAGCGCCCUGUGCGACCACUGGGAUACACUGAGGAAUGCC CUGUAUGAGCGGAGAAUCUCCGAGCUGACAGGCAAGAUCACCAAGUCUGCCAA GGAGAAGGUGCAGCGCAGCCUGAAGCACGAGGAUAUCAACCUGCAGGAGAUC AUCUCUGCCGCAGGCAAGGAGCUGAGCGAGGCCUUCAAGCAGAAAACCAGCGA GAUCCUGUCCCACGCACACGCCGCCCUGGAUCAGCCACUGCCUACAACCCUGA AGAAGCAGGAGGAGAAGGAGAUCCUGAAGUCUCAGCUGGACAGCCUGCUGGG CCUGUAC C AC CUGCUGGACUGGUUUGC C GUGGAUGAGUC C AAC GAGGUGGAC C CCGAGUUCUCUGCCCGGCUGACCGGCAUCAAGCUGGAGAUGGAGCCUUCUCUG AGCUUCUACAACAAGGCCAGAAAUUAUGCCACCAAGAAGCCCUACUCCGUGGA GAAGUUCAAGCUGAACUUUCAGCGGCCUACACUGGCCUCLIGGCUGGGACGLIGA AUAAGGAGAAGAACAAUGGCGCCAUCCUGUUUGUGAAGAACGGCCUGUACUA UCUGGGCAUCAUGCCAAAGCAGAAGGGCAGGUAUAAGGCCCUGAGCUUCGAGC CCACAGAGAAAACCAGCGAGGGCUUUGAUAAGAUGUACUAUGACUACUUCCCU GAUGCCGCCAAGAUGAUCCCAAAGUGCAGCACCCAGCUGAAGGCCGUGACAGC CCACUUUCAGACCCACACAACCCCCAUCCUGCUGUCCAACAAUUUCAUCGAGC CUCUGGAGAUCACAAAGGAGAUCUACGACCUGAACAAUCCUGAGAAGGAGCCA AAGAAGUUUCAGACAGCCUACGCCAAGAAAACCGGCGACCAGAAGGGCUACAG

[0851] AGAGGCCCUGUGCAAGUGGAUCGACUUCACAAGGGAUUUUCUGUCCAAGUAU ACCAAGACAACCUCUAUCGAUCUGUCUAGCCUGCGGCCAUCCUCUCAGUAUAA GGACCUGGGCGAGUACUAUGCCGAGCUGAAUCCCCUGCUGUACCACAUCAGCU UCCAGAGAAUCGCCGAGAAGGAGAUCAUGGAUGCCGUGGAGACAGGCAAGCU GUACCUGUUCCAGAUCUAUAACAAGGACUUUGCCAAGGGCCACCACGGCAAGC CUAAUCUGCACACACUGUAUUGGACCGGCCUGUUUUCUCCAGAGAACCUGGCC AAGACAAGCAUCAAGCUGAAUGGCCAGGCCGAGCUGUUCUACCGCCCUAAGUC CAGGAUGAAGAGGAUGGCAGCUCGGCUGGGAGAGAAGAUGCUGAACAAGAAG CUGAAGGAUCAGAAAACCCCAAUCCCCGACACCCUGUACCAGGAGCUGUACGA CUAUGUGAAUCACAGACUGUCCCACGACCUGUCUGAUGAGGCCAGGGCCCUGC UGCCCAACGUGAUCACCAAGGAGGUGUCUCACGAGAUCAUCAAGGAUAGGCGC UUUACCAGCGACAAGUUCUUGUUCCACGUGCCUAUCACACUGAACUAUCAGGC

[0852] CGCCAAUUCCCCAUCUAAGUUCAACCAGAGGGUGAAUGCCUACCUGAAGGAGC

[0853] ACCCCGAGACACCUAUCAUCGGCAUCGAUCGGGGCGAGAGAAACCUGAUCUAU

[0854] AUCACAGUGAUCGACUCCACCGGCAAGAUCCUGGAGCAGCGGAGCCUGAACAC

[0855] CAUCCAGCAGUUUGAUUACCAGAAGAAGCUGGACAACAGGGAGAAGGAGAGG

[0856] GUGGCAGCAAGGCAGGCCUGGUCUGUGGUGGGCACAAUCAAGGAUCUGAAGC

[0857] AGGGCUAUCUGAGCCAGGUCAUCCACGAGAUCGUGGACCUGAUGAUCCACUAC

[0858] CAGGCCGUGGUGGUGCUGGAGAACCUGAAUUUCGGCUUUAAGAGCAAGAGGA

[0859] CCGGCAUCGCCGAGAAGGCCGUGUACCAGCAGUUCGAGAAGAUGCUGAUCGAU

[0860] AAGCUGAAUUGCCUGGUGCUGAAGGACUAUCCAGCAGAGAAAGUGGGAGGCG

[0861] UGCUGAACCCAUACCAGCUGACAGACCAGUUCACCUCCUUUGCCAAGAUGGGC

[0862] ACCCAGUCUGGCUUCCUGUUUUACGUGCCUGCCCCAUAUACAUCUAAGAUCGA

[0863] UCCCCUGACCGGCUUCGUGGACCCCUUCGUGUGGAAAACCAUCAAGAAUCACG

[0864] AGAGCCGCAAGCACUUCCUGGAGGGCUUCGACUUUCUGCACUACGACGUGAAA

[0865] ACCGGCGACUUCAUCCUGCACUUUAAGAUGAACAGAAAUCUGUCCUUCCAGAG

[0866] GGGCCUGCCCGGCUUUAUGCCUGCAUGGGAUAUCGUGUUCGAGAAGAACGAG

[0867] ACACAGUUUGACGCCAAGGGCACCCCUUUCAUCGCCGGCAAGAGAAUCGUGCC

[0868] AGUGAUCGAGAAUCACAGAUUCACCGGCAGAUACCGGGACCUGUAUCCUGCCA

[0869] ACGAGCUGAUCGCCCUGCUGGAGGAGAAGGGCAUCGUGUUCAGGGAUGGCUCC

[0870] AACAUCCUGCCAAAGCUGCUGGAGAAUGACGAUUCUCACGCCAUCGACACCAU

[0871] GGUGGCCCUGAUCCGCAGCGUGCUGCAGAUGCGGAACUCCAAUGCCGCCACAG

[0872] GCGAGGACUAUAUCAACAGCCCCGUGCGCGAUCUGAAUGGCGUGUGCUUCGAC

[0873] UCCCGGUUUCAGAACCCAGAGUGGCCCAUGGACGCCGAUGCCAAUGGCGCCUA

[0874] CCACAUCGCCCUGAAGGGCCAGCUGCUGCUGAAUCACCUGAAGGAGAGCAAGG

[0875] AUCUGAAGCUGCAGAACGGCAUCUCCAAUCAGGACUGGCUGGCCUACAUCCAG

[0876] GAGCUGCGCAACGGCGGAUCCCCUGCUGCUAAACGUGUUAAGCUUGAUGGGGG

[0877] UAGCCCGGCAGCCAAGAGAGUCAAACUCGACUAGAUGGUUUAUAUUGCGGCCG

[0878] CUUAAUUAAGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUC

[0879] UCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUAGGAAGUCUA

[0880] G [SEQ ID NO: 434] mRNA #8

[0881] (Uridine Modifications = N1 -Methyl-Pseudo UTP) AGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCA UGACACAGUUCGAGGGCUUUACCAACCUGUAUCAGGUGAGCAAGACACUGCGG UUUGAGCUGAUCCCACAGGGCAAGACCCUGAAGCACAUCCAGGAGCAGGGCUU CAUCGAGGAGGACAAGGCCCGCAAUGAUCACUACAAGGAGCUGAAGCCCAUCA UCGAUCGGAUCUACAAGACCUAUGCCGACCAGUGCCUGCAGCUGGUGCAGCUG GAUUGGGAGAACCUGAGCGCCGCCAUCGACUCCUAUAGAAAGGAGAAAACCGA GGAGACAAGGAACGCCCUGAUCGAGGAGCAGGCCACAUAUCGCAAUGCCAUCC ACGACU ACUUC AUC GGCCGGAC AGAC A ACCUGACC GAUGCC AUC A AU A AGAGA CACGCCGAGAUCUACAAGGGCCUGUUCAAGGCCGAGCUGUUUAAUGGCAAGGU GCUGAAGCAGCUGGGCACCGUGACCACAACCGAGCACGAGAACGCCCUGCUGC GGAGCUUCGACAAGUUUACAACCUACUUCUCCGGCUUUUAUGAAAACAGGAA GAACGUGUUCAGCGCCGAGGAUAUCAGCACAGCCAUCCCACACCGCAUCGUGC AGGACAACUUCCCCAAGUUUAAGGAGAAUUGUCACAUCUUCACACGCCUGAUC ACCGCCGUGCCCAGCCUGCGGGAGCACUUUGAGAACGUGAAGAAGGCCAUCGG CAUCUUCGUGAGCACCUCCAUCGAGGAGGUGUUUUCCUUCCCUUUUUAUAACC AGCUGCUGACACAGACCCAGAUCGACCUGUAUAACCAGCUGCUGGGAGGAAUC UCUCGGGAGGCAGGCACCGAGAAGAUCAAGGGCCUGAACGAGGUGCUGAAUC UGGCCAUCCAGAAGAAUGAUGAGACAGCCCACAUCAUCGCCUCCCUGCCACAC AGAUUCAUCCCCCUGUUUAAGCAGAUCCUGUCCGAUAGGAACACCCUGUCUUU CAUCCUGGAGGAGUUUAAGAGCGACGAGGAAGUGAUCCAGUCCUUCUGCAAG UACAAGACACUGCUGAGAAACGAGAACGUGCUGGAGACAGCCGAGGCCCUGUU UAACGAGCUGAACAGCAUCGACCUGACACACAUCUUCAUCAGCCACAAGAAGC UGGAGACAAUCAGCAGCGCCCUGUGCGACCACUGGGAUACACUGAGGAAUGCC CUGUAUGAGCGGAGAAUCUCCGAGCUGACAGGCAAGAUCACCAAGUCUGCCAA GGAGAAGGUGCAGCGCAGCCUGAAGCACGAGGAUAUCAACCUGCAGGAGAUC AUCUCUGCCGCAGGCAAGGAGCUGAGCGAGGCCUUCAAGCAGAAAACCAGCGA GAUCCUGUCCCACGCACACGCCGCCCUGGAUCAGCCACUGCCUACAACCCUGA AGAAGCAGGAGGAGAAGGAGAUCCUGAAGUCUCAGCUGGACAGCCUGCUGGG

[0882] CCUGUACCACCUGCUGGACUGGUUUGCCGUGGAUGAGUCCAACGAGGUGGACC CCGAGUUCUCUGCCCGGCUGACCGGCAUCAAGCUGGAGAUGGAGCCUUCUCUG AGCUUCUACAACAAGGCCAGAAAUUAUGCCACCAAGAAGCCCUACUCCGUGGA GAAGUUCAAGCUGAACUUUCAGCGGCCUACACUGGCCUCUGGCUGGGACGUGA AUAAGGAGAAGAACAAUGGCGCCAUCCUGUUUGUGAAGAACGGCCUGUACUA UCUGGGCAUCAUGCCAAAGCAGAAGGGCAGGUAUAAGGCCCUGAGCUUCGAGC CCACAGAGAAAACCAGCGAGGGCUUUGAUAAGAUGUACUAUGACUACUUCCCU GAUGCCGCCAAGAUGAUCCCAAAGUGCAGCACCCAGCUGAAGGCCGUGACAGC CCACUUUCAGACCCACACAACCCCCAUCCUGCUGUCCAACAAUUUCAUCGAGC

[0883] CUCUGGAGAUCACAAAGGAGAUCUACGACCUGAACAAUCCUGAGAAGGAGCCA AAGAAGUUUCAGACAGCCUACGCCAAGAAAACCGGCGACCAGAAGGGCUACAG AGAGGCCCUGUGCAAGUGGAUCGACUUCACAAGGGAUUUUCUGUCCAAGUAU ACCAAGACAACCUCUAUCGAUCUGUCUAGCCUGCGGCCAUCCUCUCAGUAUAA GGACCUGGGCGAGUACUAUGCCGAGCUGAAUCCCCUGCUGUACCACAUCAGCU UCCAGAGAAUCGCCGAGAAGGAGAUCAUGGAUGCCGUGGAGACAGGCAAGCU GUACCUGUUCCAGAUCUAUAACAAGGACUUUGCCAAGGGCCACCACGGCAAGC CUAAUCUGCACACACUGUAUUGGACCGGCCUGUUUUCUCCAGAGAACCUGGCC AAGACAAGCAUCAAGCUGAAUGGCCAGGCCGAGCUGUUCUACCGCCCUAAGUC CAGGAUGAAGAGGAUGGCAGCUCGGCUGGGAGAGAAGAUGCUGAACAAGAAG CUGAAGGAUCAGAAAACCCCAAUCCCCGACACCCUGUACCAGGAGCUGUACGA CUAUGUGAAUCACAGACUGUCCCACGACCUGUCUGAUGAGGCCAGGGCCCUGC UGCCCAACGUGAUCACCAAGGAGGUGUCUCACGAGAUCALICAAGGAUAGGCGC UUUACCAGCGACAAGUUCUUGUUCCACGUGCCUAUCACACUGAACUAUCAGGC CGC C AAUUC CCC AUCUAAGUUC AAC C AGAGGGUGAAUGCCUAC CUGAAGGAGC ACCCCGAGACACCUAUCAUCGGCAUCGAUCGGGGCGAGAGAAACCUGAUCUAU AUCACAGUGAUCGACUCCACCGGCAAGAUCCUGGAGCAGCGGAGCCUGAACAC CAUCCAGCAGUUUGAUUACCAGAAGAAGCUGGACAACAGGGAGAAGGAGAGG

[0884] GUGGCAGCAAGGCAGGCCUGGUCUGUGGUGGGCACAAUCAAGGAUCUGAAGC AGGGCUAUCUGAGCCAGGUCAUCCACGAGAUCGUGGACCUGAUGAUCCACUAC CAGGCCGUGGUGGUGCUGGAGAACCUGAAUUUCGGCUUUAAGAGCAAGAGGA

[0885] CCGGCAUCGCCGAGAAGGCCGUGUACCAGCAGUUCGAGAAGAUGCUGAUCGAU AAGCUGAAUUGCCUGGUGCUGAAGGACUAUCCAGCAGAGAAAGUGGGAGGCG UGCUGAACCCAUACCAGCUGACAGACCAGUUCACCUCCUUUGCCAAGAUGGGC ACCCAGUCUGGCUUCCUGUUUUACGUGCCUGCCCCAUAUACAUCUAAGAUCGA UCCCCUGACCGGCUUCGUGGACCCCUUCGUGUGGAAAACCAUCAAGAAUCACG AGAGCCGCAAGCACUUCCUGGAGGGCUUCGACUUUCUGCACUACGACGUGAAA ACCGGCGACUUCAUCCUGCACUUUAAGAUGAACAGAAAUCUGUCCUUCCAGAG GGGCCUGCCCGGCUUUAUGCCUGCAUGGGAUAUCGUGUUCGAGAAGAACGAG ACACAGUUUGACGCCAAGGGCACCCCUUUCAUCGCCGGCAAGAGAAUCGUGCC AGUGAUCGAGAAUCACAGAUUCACCGGCAGAUACCGGGACCUGUAUCCUGCCA ACGAGCUGAUCGCCCUGCUGGAGGAGAAGGGCAUCGUGUUCAGGGAUGGCUCC

[0886] AACAUCCUGCCAAAGCUGCUGGAGAAUGACGAUUCUCACGCCAUCGACACCAU

[0887] GGUGGCCCUGAUCCGCAGCGUGCUGCAGAUGCGGAACUCCAAUGCCGCCACAG

[0888] GCGAGGACUAUAUCAACAGCCCCGUGCGCGAUCUGAAUGGCGUGUGCUUCGAC

[0889] UCCCGGUUUCAGAACCCAGAGUGGCCCAUGGACGCCGAUGCCAAUGGCGCCUA

[0890] CCACAUCGCCCUGAAGGGCCAGCUGCUGCUGAAUCACCUGAAGGAGAGCAAGG

[0891] AUCUGAAGCUGCAGAACGGCAUCUCCAAUCAGGACUGGCUGGCCUACAUCCAG

[0892] GAGCUGCGCAACGGCGGAUCCCCUGCUGCUAAACGUGUUAAGCUUGAUGGGGG

[0893] UAGCCCGGCAGCCAAGAGAGUCAAACUCGACUAGAUGGUUUAUAUUGCGGCCG

[0894] CUUAAUUAAGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUC

[0895] UCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUAGGAAGUCUA

[0896] G [SEQ ID NO: 435] mRNA #9

[0897] (Uridine Modifications = N1 -Methyl-Pseudo UTP)

[0898] AUGACACAGUUCGAGGGCUUUACCAACCUGUAUCAGGUGAGCAAGACA

[0899] CUGCGGUUUGAGCUGAUCCCACAGGGCAAGACCCUGAAGCACAUCCAGGAGCA

[0900] GGGCUUCAUCGAGGAGGACAAGGCCCGCAAUGAUCACUACAAGGAGCUGAAGC

[0901] CCAUCAUCGAUCGGAUCUACAAGACCUAUGCCGACCAGUGCCUGCAGCUGGUG

[0902] CAGCUGGAUUGGGAGAACCUGAGCGCCGCCAUCGACUCCUAUAGAAAGGAGAA

[0903] AACCGAGGAGACAAGGAACGCCCUGAUCGAGGAGCAGGCCACAUAUCGCAAUG

[0904] CCAUCCACGACUACUUCAUCGGCCGGACAGACAACCUGACCGAUGCCAUCAAU

[0905] AAGAGACACGCCGAGAUCUACAAGGGCCUGUUCAAGGCCGAGCUGUUUAAUG

[0906] GCAAGGUGCUGAAGCAGCUGGGCACCGUGACCACAACCGAGCACGAGAACGCC

[0907] CUGCUGCGGAGCUUCGACAAGUUUACAACCUACUUCUCCGGCUUUUAUGAAAA

[0908] CAGGAAGAACGUGUUCAGCGCCGAGGAUAUCAGCACAGCCAUCCCACACCGCA

[0909] UCGUGCAGGACAACUUCCCCAAGUUUAAGGAGAAUUGUCACAUCUUCACACGC

[0910] CUGAUCACCGCCGUGCCCAGCCUGCGGGAGCACUUUGAGAACGUGAAGAAGGC

[0911] CAUCGGCAUCUUCGUGAGCACCUCCAUCGAGGAGGUGUUUUCCUUCCCUUUUU

[0912] AUAACCAGCUGCUGACACAGACCCAGAUCGACCUGUAUAACCAGCUGCUGGGA

[0913] GGAAUCUCUCGGGAGGCAGGCACCGAGAAGAUCAAGGGCCUGAACGAGGUGC

[0914] UGAAUCUGGCCAUCCAGAAGAAUGAUGAGACAGCCCACAUCAUCGCCUCCCUG

[0915] CCACACAGAUUCAUCCCCCUGUUUAAGCAGAUCCUGUCCGAUAGGAACACCCU

[0916] GUCUUUCAUCCUGGAGGAGUUUAAGAGCGACGAGGAAGUGAUCCAGUCCUUC UGCAAGUACAAGACACUGCUGAGAAACGAGAACGUGCUGGAGACAGCCGAGG

[0917] CCCUGUUUAACGAGCUGAACAGCAUCGACCUGACACACAUCUUCAUCAGCCAC

[0918] AAGAAGCUGGAGACAAUCAGCAGCGCCCUGUGCGACCACUGGGAUACACUGAG

[0919] GAAUGCCCUGUAUGAGCGGAGAAUCUCCGAGCUGACAGGCAAGAUCACCAAGU

[0920] CUGCCAAGGAGAAGGUGCAGCGCAGCCUGAAGCACGAGGAUAUCAACCUGCAG

[0921] GAGAUCAUCUCUGCCGCAGGCAAGGAGCUGAGCGAGGCCUUCAAGCAGAAAAC

[0922] CAGCGAGAUCCUGUCCCACGCACACGCCGCCCUGGAUCAGCCACUGCCUACAA

[0923] CCCUGAAGAAGCAGGAGGAGAAGGAGAUCCUGAAGUCUCAGCUGGACAGCCU

[0924] GCUGGGCCUGUACCACCUGCUGGACUGGUUUGCCGUGGAUGAGUCCAACGAGG

[0925] UGGACCCCGAGUUCUCUGCCCGGCUGACCGGCAUCAAGCUGGAGAUGGAGCCU

[0926] UCUCUGAGCUUCUACAACAAGGCCAGAAAUUAUGCCACCAAGAAGCCCUACUC

[0927] CGUGGAGAAGUUCAAGCUGAACUUUCAGCGGCCUACACUGGCCUCUGGCUGGG

[0928] ACGUGAAUAAGGAGAAGAACAAUGGCGCCAUCCUGUUUGUGAAGAACGGCCU

[0929] GUACUAUCUGGGCAUCAUGCCAAAGCAGAAGGGCAGGUAUAAGGCCCUGAGC

[0930] UUCGAGCCCACAGAGAAAACCAGCGAGGGCUUUGAUAAGAUGUACUAUGACU

[0931] ACUUCCCUGAUGCCGCCAAGAUGAUCCCAAAGUGCAGCACCCAGCUGAAGGCC

[0932] GUGACAGCCCACUUUCAGACCCACACAACCCCCAUCCUGCUGUCCAACAAUUU

[0933] CAUCGAGCCUCUGGAGAUCACAAAGGAGAUCUACGACCUGAACAAUCCUGAGA

[0934] AGGAGCCAAAGAAGUUUCAGACAGCCUACGCCAAGAAAACCGGCGACCAGAAG

[0935] GGCUACAGAGAGGCCCUGUGCAAGUGGAUCGACUUCACAAGGGAUUUUCUGU

[0936] CCAAGUAUACCAAGACAACCUCUAUCGAUCUGUCUAGCCUGCGGCCAUCCUCU

[0937] CAGUAUAAGGACCUGGGCGAGUACUAUGCCGAGCUGAAUCCCCUGCUGUACCA

[0938] CAUCAGCUUCCAGAGAAUCGCCGAGAAGGAGAUCAUGGAUGCCGUGGAGACA

[0939] GGCAAGCUGUACCUGUUCCAGAUCUAUAACAAGGACUUUGCCAAGGGCCACCA

[0940] CGGCAAGCCUAAUCUGCACACACUGUAUUGGACCGGCCUGUUUUCUCCAGAGA

[0941] ACCUGGCCAAGACAAGCAUCAAGCUGAAUGGCCAGGCCGAGCUGUUCUACCGC

[0942] CCUAAGUCCAGGAUGAAGAGGAUGGCAGCUCGGCUGGGAGAGAAGAUGCUGA

[0943] ACAAGAAGCUGAAGGAUCAGAAAACCCCAAUCCCCGACACCCUGUACCAGGAG

[0944] CUGUACGACUAUGUGAAUCACAGACUGUCCCACGACCUGUCUGAUGAGGCCAG

[0945] GGCCCUGCUGCCCAACGUGAUCACCAAGGAGGUGUCUCACGAGAUCAUCAAGG

[0946] AUAGGCGCUUUACCAGCGACAAGUUCUUGUUCCACGUGCCUAUCACACUGAAC

[0947] UAUCAGGCCGCCAAUUCCCCAUCUAAGUUCAACCAGAGGGUGAAUGCCUACCU

[0948] GAAGGAGCACCCCGAGACACCUAUCAUCGGCAUCGAUCGGGGCGAGAGAAACC

[0949] UGAUCUAUAUCACAGUGAUCGACUCCACCGGCAAGAUCCUGGAGCAGCGGAGC CUGAACACCAUCCAGCAGUUUGAUUACCAGAAGAAGCUGGACAACAGGGAGA

[0950] AGGAGAGGGUGGCAGCAAGGCAGGCCUGGUCUGUGGUGGGCACAAUCAAGGA

[0951] UCUGAAGCAGGGCUAUCUGAGCCAGGUCAUCCACGAGAUCGUGGACCUGAUGA

[0952] UCCACUACCAGGCCGUGGUGGUGCUGGAGAACCUGAAUUUCGGCUUUAAGAGC

[0953] AAGAGGACCGGCAUCGCCGAGAAGGCCGUGUACCAGCAGUUCGAGAAGAUGCU

[0954] GAUCGAUAAGCUGAAUUGCCUGGUGCUGAAGGACUAUCCAGCAGAGAAAGUG

[0955] GGAGGCGUGCUGAACCCAUACCAGCUGACAGACCAGUUCACCUCCUUUGCCAA

[0956] GAUGGGCACCCAGUCUGGCUUCCUGUUUUACGUGCCUGCCCCAUAUACAUCUA

[0957] AGAUCGAUCCCCUGACCGGCUUCGUGGACCCCUUCGUGUGGAAAACCAUCAAG

[0958] AAUCACGAGAGCCGCAAGCACUUCCUGGAGGGCUUCGACUUUCUGCACUACGA

[0959] CGUGAAAACCGGCGACUUCAUCCUGCACUUUAAGAUGAACAGAAAUCUGUCCU

[0960] UCCAGAGGGGCCUGCCCGGCUUUAUGCCUGCAUGGGAUAUCGUGUUCGAGAAG

[0961] AACGAGACACAGUUUGACGCCAAGGGCACCCCUUUCAUCGCCGGCAAGAGAAU

[0962] CGUGCCAGUGAUCGAGAAUCACAGAUUCACCGGCAGAUACCGGGACCUGUAUC

[0963] CUGCCAACGAGCUGAUCGCCCUGCUGGAGGAGAAGGGCAUCGUGUUCAGGGAU

[0964] GGCUCCAACAUCCUGCCAAAGCUGCUGGAGAAUGACGAUUCUCACGCCAUCGA

[0965] CACCAUGGUGGCCCUGAUCCGCAGCGUGCUGCAGAUGCGGAACUCCAAUGCCG

[0966] CCACAGGCGAGGACUAUAUCAACAGCCCCGUGCGCGAUCUGAAUGGCGUGUGC

[0967] UUCGACUCCCGGUUUCAGAACCCAGAGUGGCCCAUGGACGCCGAUGCCAAUGG

[0968] CGCCUACCACAUCGCCCUGAAGGGCCAGCUGCUGCUGAAUCACCUGAAGGAGA

[0969] GCAAGGAUCUGAAGCUGCAGAACGGCAUCUCCAAUCAGGACUGGCUGGCCUAC

[0970] AUCCAGGAGCUGCGCAACGGCGGAUCCCCUGCUGCUAAACGUGUUAAGCUUGA

[0971] UGGGGGUAGCCCGGCAGCCAAGAGAGUCAAACUCGAC [SEQ ID NO: 436] mRNA #10

[0972] UGACACAGUUCGAGGGCUUUACCAACCUGUAUCAGGUGAGCAAGACAC

[0973] UGCGGUUUGAGCUGAUCCCACAGGGCAAGACCCUGAAGCACAUCCAGGAGCAG

[0974] GGCUUCAUCGAGGAGGACAAGGCCCGCAAUGAUCACUACAAGGAGCUGAAGCC

[0975] CAUCAUCGAUCGGAUCUACAAGACCUAUGCCGACCAGUGCCUGCAGCUGGUGC

[0976] AGCUGGAUUGGGAGAACCUGAGCGCCGCCAUCGACUCCUAUAGAAAGGAGAA

[0977] AACCGAGGAGACAAGGAACGCCCUGAUCGAGGAGCAGGCCACAUAUCGCAAUG

[0978] CCAUCCACGACUACUUCAUCGGCCGGACAGACAACCUGACCGAUGCCAUCAAU

[0979] AAGAGACACGCCGAGAUCUACAAGGGCCUGUUCAAGGCCGAGCUGUUUAAUG

[0980] GCAAGGUGCUGAAGCAGCUGGGCACCGUGACCACAACCGAGCACGAGAACGCC CUGCUGCGGAGCUUCGACAAGUUUACAACCUACUUCUCCGGCUUUUAUGAAAA CAGGAAGAACGUGUUCAGCGCCGAGGAUAUCAGCACAGCCAUCCCACACCGCA UCGUGCAGGACAACUUCCCCAAGUUUAAGGAGAAUUGUCACAUCUUCACACGC CUGAUCACCGCCGUGCCCAGCCUGCGGGAGCACUUUGAGAACGUGAAGAAGGC CAUCGGCAUCUUCGUGAGCACCUCCAUCGAGGAGGUGUUUUCCUUCCCUUUUU AUAACCAGCUGCUGACACAGACCCAGAUCGACCUGUAUAACCAGCUGCUGGGA GGAAUCUCUCGGGAGGCAGGCACCGAGAAGAUCAAGGGCCUGAACGAGGUGC UGAAUCUGGCCAUCCAGAAGAAUGAUGAGACAGCCCACAUCAUCGCCUCCCUG CCACACAGAUUCAUCCCCCUGUUUAAGCAGAUCCUGUCCGAUAGGAACACCCU GUCUUUCAUCCUGGAGGAGUUUAAGAGCGACGAGGAAGUGAUCCAGUCCUUC UGCAAGUACAAGACACUGCUGAGAAACGAGAACGUGCUGGAGACAGCCGAGG CCCUGUUUAACGAGCUGAACAGCAUCGACCUGACACACAUCUUCAUCAGCCAC AAGAAGCUGGAGACAAUCAGCAGCGCCCUGUGCGACCACUGGGAUACACUGAG GAAUGCCCUGUAUGAGCGGAGAAUCUCCGAGCUGACAGGCAAGAUCACCAAGU CUGCCAAGGAGAAGGUGCAGCGCAGCCUGAAGCACGAGGAUAUCAACCUGCAG GAGAU C AUC UC UGCCGC AGGC AAGGAGC U GAGCGAGGCC UUC AAGC AGAAAAC CAGCGAGAUCCUGUCCCACGCACACGCCGCCCUGGAUCAGCCACUGCCUACAA CCCUGAAGAAGCAGGAGGAGAAGGAGAUCCUGAAGUCUCAGCUGGACAGCCU GCUGGGCCUGUACCACCUGCUGGACUGGUUUGCCGUGGAUGAGUCCAACGAGG UGGACCCCGAGUUCUCUGCCCGGCUGACCGGCAUCAAGCUGGAGAUGGAGCCU

[0981] UCUCUGAGCUUCUACAACAAGGCCAGAAAUUAUGCCACCAAGAAGCCCUACUC CGUGGAGAAGUUCAAGCUGAACUUUCAGCGGCCUACACUGGCCUCUGGCUGGG ACGUGAAUAAGGAGAAGAACAAUGGCGCCAUCCUGUUUGUGAAGAACGGCCU GUACUAUCUGGGCAUCAUGCCAAAGCAGAAGGGCAGGUAUAAGGCCCUGAGC UUCGAGCCCACAGAGAAAACCAGCGAGGGCUUUGAUAAGAUGUACUAUGACU ACUUCCCUGAUGCCGCCAAGAUGAUCCCAAAGUGCAGCACCCAGCUGAAGGCC GUGACAGCCCACUUUCAGACCCACACAACCCCCAUCCUGCUGUCCAACAAUUU CAUCGAGCCUCUGGAGAUCACAAAGGAGAUCUACGACCUGAACAAUCCUGAGA AGGAGCCAAAGAAGUUUCAGACAGCCUACGCCAAGAAAACCGGCGACCAGAAG GGCUACAGAGAGGCCCUGUGCAAGUGGAUCGACUUCACAAGGGAUUUUCUGU CCAAGUAUACCAAGACAACCUCUAUCGAUCUGUCUAGCCUGCGGCCAUCCUCU CAGUAUAAGGACCUGGGCGAGUACUAUGCCGAGCUGAAUCCCCUGCUGUACCA CAUCAGCUUCCAGAGAAUCGCCGAGAAGGAGAUCAUGGAUGCCGUGGAGACA GGCAAGCUGUACCUGUUCCAGAUCUAUAACAAGGACUUUGCCAAGGGCCACCA CGGCAAGCCUAAUCUGCACACACUGUAUUGGACCGGCCUGUUUUCUCCAGAGA

[0982] ACCUGGCCAAGACAAGCAUCAAGCUGAAUGGCCAGGCCGAGCUGUUCUACCGC

[0983] CCUAAGUCCAGGAUGAAGAGGAUGGCAGCUCGGCUGGGAGAGAAGAUGCUGA

[0984] ACAAGAAGCUGAAGGAUCAGAAAACCCCAAUCCCCGACACCCUGUACCAGGAG

[0985] CUGUACGACUAUGUGAAUCACAGACUGUCCCACGACCUGUCUGAUGAGGCCAG

[0986] GGCCCUGCUGCCCAACGUGAUCACCAAGGAGGUGUCUCACGAGAUCAUCAAGG

[0987] AUAGGCGCUUUACCAGCGACAAGUUCUUGUUCCACGUGCCUAUCACACUGAAC

[0988] UAUCAGGCCGCCAAUUCCCCAUCUAAGUUCAACCAGAGGGUGAAUGCCUACCU

[0989] GAAGGAGCACCCCGAGACACCUAUCAUCGGCAUCGAUCGGGGCGAGAGAAACC

[0990] UGAUCUAUAUCACAGUGAUCGACUCCACCGGCAAGAUCCUGGAGCAGCGGAGC

[0991] CUGAACACCAUCCAGCAGUUUGAUUACCAGAAGAAGCUGGACAACAGGGAGA

[0992] AGGAGAGGGUGGCAGCAAGGCAGGCCUGGUCUGUGGUGGGCACAAUCAAGGA

[0993] UCUGAAGCAGGGCUAUCUGAGCCAGGUCAUCCACGAGAUCGUGGACCUGAUGA

[0994] UCCACUACCAGGCCGUGGUGGUGCUGGAGAACCUGAAUUUCGGCUUUAAGAGC

[0995] AAGAGGACCGGCAUCGCCGAGAAGGCCGUGUACCAGCAGUUCGAGAAGAUGCU

[0996] GAUCGAUAAGCUGAAUUGCCUGGUGCUGAAGGACUAUCCAGCAGAGAAAGUG

[0997] GGAGGCGUGCUGAACCCAUACCAGCUGACAGACCAGUUCACCUCCUUUGCCAA

[0998] GAUGGGCACCCAGUCUGGCUUCCUGUUUUACGUGCCUGCCCCAUAUACAUCUA

[0999] AGAUCGAUCCCCUGACCGGCUUCGUGGACCCCUUCGUGUGGAAAACCAUCAAG

[1000] AAUCACGAGAGCCGCAAGCACUUCCUGGAGGGCUUCGACUUUCUGCACUACGA

[1001] CGUGAAAACCGGCGACUUCAUCCUGCACUUUAAGAUGAACAGAAAUCUGUCCU

[1002] UCCAGAGGGGCCUGCCCGGCUUUAUGCCUGCAUGGGAUAUCGUGUUCGAGAAG

[1003] AACGAGACACAGUUUGACGCCAAGGGCACCCCUUUCAUCGCCGGCAAGAGAAU

[1004] CGUGCCAGUGAUCGAGAAUCACAGAUUCACCGGCAGAUACCGGGACCUGUAUC

[1005] CUGCCAACGAGCUGAUCGCCCUGCUGGAGGAGAAGGGCAUCGUGUUCAGGGAU

[1006] GGCUCCAACAUCCUGCCAAAGCUGCUGGAGAAUGACGAUUCUCACGCCAUCGA

[1007] CACCAUGGUGGCCCUGAUCCGCAGCGUGCUGCAGAUGCGGAACUCCAAUGCCG

[1008] CCACAGGCGAGGACUAUAUCAACAGCCCCGUGCGCGAUCUGAAUGGCGUGUGC

[1009] UUCGACUCCCGGUUUCAGAACCCAGAGUGGCCCAUGGACGCCGAUGCCAAUGG

[1010] CGCCUACCACAUCGCCCUGAAGGGCCAGCUGCUGCUGAAUCACCUGAAGGAGA

[1011] GCAAGGAUCUGAAGCUGCAGAACGGCAUCUCCAAUCAGGACUGGCUGGCCUAC

[1012] AUCCAGGAGCUGCGCAACGGCGGAUCCCCUGCUGCUAAACGUGUUAAGCUUGA

[1013] UGGGGGUAGCCCGGCAGCCAAGAGAGUCAAACUCGAC [SEQ ID NO: 437] 5.5 Functional analysis of candidate molecules

[1014] Candidate RNA-guided nucleases, gRNAs, and complexes thereof, can be evaluated by standard methods known in the art. See, e.g, Cotta-Ramusino. The stability of RNP complexes can be evaluated by differential scanning fluorimetry, as described below.

[1015] 5.5.1 Differential Scanning Fluorimetry (DSF)

[1016] The thermostability of ribonucleoprotein (RNP) complexes comprising gRNAs and RNA-guided nucleases can be measured via DSF. The DSF technique measures the thermostability of a protein, which can increase under favorable conditions such as the addition of a binding RNA molecule, e.g., a gRNA.

[1017] A DSF assay can be performed according to any suitable protocol, and can be employed in any suitable setting, including without limitation (a) testing different conditions (e.g. different stoichiometric ratios of gRNA: RNA-guided nuclease protein, different buffer solutions, etc.) to identify optimal conditions for RNP complex formation; and (b) testing modifications (e.g. chemical modifications, alterations of sequence, etc.) of an RNA-guided nuclease and / or a gRNA to identify those modifications that improve RNP complex formation or stability. One readout of a DSF assay is a shift in melting temperature of the RNP complex; a relatively high shift suggests that the RNP complex is more stable (and can thus have greater activity or more favorable kinetics of formation, kinetics of degradation, or another functional characteristic) relative to a reference RNP complex characterized by a lower shift. When the DSF assay is deployed as a screening tool, a threshold melting temperature shift can be specified, so that the output is one or more RNP complexes having a melting temperature shift at or above the threshold. For instance, the threshold can be 5-10°C (e.g, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C) or more, and the output can be one or more RNP complexes characterized by a melting temperature shift greater than or equal to the threshold.

[1018] Two non-limiting examples of DSF assay conditions are set forth below:

[1019] To determine the best solution to form RNP complexes, a fixed concentration (e.g, 2 pM) of RNA-guided nuclease (e.g., Cas9 or Casl2a) in water+lOx SYPRO Orange® (Life Technologies cat#S-6650) is dispensed into a 384 well plate. An equimolar amount of gRNA diluted in solutions with varied pH and salt is then added. After incubating at room temperature for 10’and brief centrifugation to remove any bubbles, a Bio-Rad CFX384™ Real-Time System Cl 000 Touch™ Thermal Cycler with the Bio-Rad CFX Manager software is used to run a gradient from 20°C to 90°C with a 1°C increase in temperature every 10 seconds. The second assay involves mixing various concentrations of gRNA with a fixed concentration (e.g.. 2 pM) of RNA-guided nuclease (e.g., Cas9 or Cas12a) in optimal buffer from assay 1 above and incubating (e.g., at RT for 10’) in a 384 well plate. An equal volume of optimal buffer + 10x SYPRO Orange® (Life Technologies cat#S-6650) is added and the plate sealed with Microseal® B adhesive (MSB-1001). Following brief centrifugation to remove any bubbles, a Bio-Rad CFX384™ Real-Time System C 1000 Touch™ Thermal Cycler with the Bio-Rad CFX Manager software is used to run a gradient from 20°C to 90°C with a 1 °C increase in temperature every 10 seconds.

[1020] 6. Genome editing strategies

[1021] The genome editing systems described above are used, in various embodiments of the present disclosure, to generate edits in (i.e., to alter) targeted regions of DNA within or obtained from a cell. Various strategies are described herein to generate particular edits, and these strategies are generally described in terms of the desired repair outcome, the number and positioning of individual edits (e.g., SSBs or DSBs), and the target sites of such edits.

[1022] Genome editing strategies that involve the formation of SSBs or DSBs are characterized by repair outcomes including: (a) deletion of all or part of a targeted region; (b) insertion into or replacement of all or part of a targeted region; or (c) interruption of all or part of a targeted region. This grouping is not intended to be limiting, or to be binding to any particular theory or model and is offered solely for economy of presentation. Skilled artisans will appreciate that the listed outcomes are not mutually exclusive and that some repairs can result in other outcomes. The description of a particular editing strategy or method should not be understood to require a particular repair outcome unless otherwise specified.

[1023] Replacement of a targeted region in certain embodiments involves the replacement of all or part of the existing sequence within the targeted region with a homologous sequence, for instance through gene correction or gene conversion, tw o repair outcomes that are mediated by HDR pathways. HDR is promoted by the use of a donor template, which can be single-stranded or double stranded, as described in greater detail below-. Single or double stranded templates can be exogenous, in which case they will promote gene correction, or they can be endogenous (e.g., a homologous sequence within the cellular genome), to promote gene conversion. Exogenous templates can have asymmetric overhangs (i.e., the portion of the template that is complementary to the site of the DSB can be offset in a 3‘ or 5‘ direction, rather than being centered within the donor template), for instance as described by Richardson et al. (Nature Biotechnology 34, 339-344 (2016), (Richardson), incorporated by reference). In instances where the template is single stranded, it can correspond to either the complementary (top) or non-complementary (bottom) strand of the targeted region.

[1024] Gene conversion and gene correction are facilitated, in some cases, by the formation of one or more nicks in or around the targeted region, as described in Ran and Cotta-Ramusino. In some cases, a dual-nickase strategy is used to form two offset SSBs that, in turn, form a single DSB having an overhang (e ., a 5’ overhang).

[1025] Interruption and / or deletion of all or part of a targeted sequence can be achieved by a variety of repair outcomes. As one example, a sequence can be deleted by simultaneously generating two or more DSBs that flank a targeted region, which is then excised when the DSBs are repaired, as is described in Maeder for the LCA10 mutation. As another example, a sequence can be interrupted by a deletion generated by formation of a double strand break with single-stranded overhangs, followed by exonucleolytic processing of the overhangs prior to repair.

[1026] One specific subset of target sequence interruptions is mediated by the formation of an indel within the targeted sequence, where the repair outcome is typically mediated by NHEJ pathways (including Alt-NHEJ). NHEJ is referred to as an "error prone" repair pathway because of its association with indel mutations. In some cases, however, a DSB is repaired by NHEJ without alteration of the sequence around it (a so-called “perfect” or “scarless” repair); this generally requires the two ends of the DSB to be perfectly ligated. Indels, meanwhile, are thought to arise from enzymatic processing of free DNA ends before they are ligated that adds and / or removes nucleotides from either or both strands of either or both free ends.

[1027] Because the enzy matic processing of free DSB ends can be stochastic in nature, indel mutations tend to be variable, occurring along a distribution, and can be influenced by a variety of factors, including the specific target site, the cell type used, the genome editing strategy used, etc. Even so, it is possible to draw limited generalizations about indel formation: deletions formed by repair of a single DSB are most commonly in the 1-50 bp range but can reach greater than 100-200 bp. Insertions formed by repair of a single DSB tend to be shorter and often comprise short duplications of the sequence immediately surrounding the break site. However, it is possible to obtain large insertions, and in these cases, the inserted sequence has often been traced to other regions of the genome or to plasmid DNA present in the cells.

[1028] Indel mutations and genome editing systems configured to produce indels are useful for interrupting target sequences, for example, when the generation of a specific final sequence is not required and / or where a frameshift mutation would be tolerated. They can also be useful in settings where particular sequences are preferred, insofar as the certain sequences desired tend to occur preferentially from the repair of an SSB or DSB at a given site. Indel mutations are also a useful tool for evaluating or screening the activity of particular genome editing systems and their components. In these and other settings, indels can be characterized by (a) their relative and absolute frequencies in the genomes of cells contacted with genome editing systems and (b) the distribution of numerical differences relative to the unedited sequence, e.g., ±1, ±2, ±3, etc. As one example, in a lead-finding setting, multiple gRNAs can be screened to identify those gRNAs that most efficiently drive cutting at a target site based on an indel readout under controlled conditions. gRNAs that produce indels at or above a threshold frequency, or that produce a particular distribution of indels, can be selected for further study and development. Indel frequency and distribution can also be useful as a readout for evaluating different genome editing system implementations or formulations and delivery methods, for instance by keeping the gRNA constant and varying certain other reaction conditions or delivery methods.

[1029] 6.1 Multiplex Strategies

[1030] While exemplary strategies discussed above have focused on repair outcomes mediated by single DSBs, genome editing systems according to this disclosure can also be employed to generate two or more DSBs, either in the same locus or in different loci. Strategies for editing that involve the formation of multiple DSBs, or SSBs, are described in, for instance, Cotta- Ramusino.

[1031] 6.2 Donor template design

[1032] Donor template design is described in detail in the literature, for instance in Cotta- Ramusino. DNA oligomer donor templates (oligodeoxynucleotides or ODNs), which can be single stranded (ssODNs) or double-stranded (dsODNs), can be used to facilitate HDR-based repair of DSBs, and are particularly useful for introducing alterations into a target DNA sequence, inserting a new sequence into the target sequence, or replacing the target sequence altogether.

[1033] Whether single-stranded or double stranded, donor templates generally comprise regions that are homologous to regions of DNA within or near (e.g., flanking or adjoining) a target sequence to be cleaved. These homologous regions are referred to here as "‘homology arms,” and are illustrated schematically below:

[1034] [5’ homology arm] — [replacement sequence] — [3’ homology arm].

[1035] The homology arms can have any suitable length (including 0 nucleotides if only one homology arm is used), and 3’ and 5’ homology arms can have the same length or can differ in length. The selection of appropriate homology arm lengths can be influenced by a variety of factors, such as the desire to avoid homologies or microhomologies with certain sequences such as Alu repeats or other very common elements. For example, a 5’ homology arm can be shortened to avoid a sequence repeat element. In other embodiments, a 3’ homology arm can be shortened to avoid a sequence repeat element. In certain embodiments, both the 5’ and the 3’ homology arms can be shortened to avoid including certain sequence repeat elements. In addition, some homology arm designs can improve the efficiency of editing or increase the frequency of a desired repair outcome. For example, Richardson et al. Nature Biotechnology 34, 339-344 (2016) (Richardson), which is incorporated by reference, found that the relative asymmetry of 3’ and 5’ homology arms of single stranded donor templates influenced repair rates and / or outcomes.

[1036] Replacement sequences in donor templates have been described elsewhere, including in Cotta-Ramusino et al. A replacement sequence can be any suitable length (including zero nucleotides, where the desired repair outcome is a deletion), and typically comprises one, two, three or more sequence modifications relative to the naturally occurring sequence within a cell in which editing is desired. One exemplary sequence modification involves the alteration of the naturally occurring sequence to repair a mutation that is related to a disease or condition of which treatment is desired. Another exemplary sequence modification involves the alteration of one or more sequences that are complementary to, or code for, the PAM sequence of the RNA-guided nuclease or the targeting domain of the gRNA(s) being used to generate an SSB or DSB, to reduce or eliminate repeated cleavage of the target site after the replacement sequence has been incorporated into the target site.

[1037] Where a linear ssODN is used, it can be configured to (i) anneal to the nicked strand of the target nucleic acid, (ii) anneal to the intact strand of the target nucleic acid, (iii) anneal to the plus strand of the target nucleic acid, and / or (iv) anneal to the minus strand of the target nucleic acid. An ssODN can have any suitable length, e.g., about, at least, or no more than 150- 200 nucleotides (e.g, 150, 160, 170, 180, 190, or 200 nucleotides).

[1038] It should be noted that a template nucleic acid can also be a nucleic acid vector, such as a viral genome or circular double stranded DNA, e.g., a plasmid. Nucleic acid vectors comprising donor templates can comprise other coding or non-coding elements. For example, a template nucleic acid can be delivered as part of a viral genome (e.g., in an AAV or lentiviral genome) that comprises certain genomic backbone elements (e.g., inverted terminal repeats, in the case of an AAV genome) and optionally comprises additional sequences coding for a gRNA and / or an RNA-guided nuclease. In certain embodiments, the donor template can be adjacent to, or flanked by. target sites recognized by one or more gRNAs, to facilitate the formation of free DSBs on one or both ends of the donor template that can participate in repair of corresponding SSBs or DSBs formed in cellular DNA using the same gRNAs. Exemplary nucleic acid vectors suitable for use as donor templates are described in Cotta-Ramusino.

[1039] Whatever format is used, a template nucleic acid can be designed to avoid undesirable sequences. In certain embodiments, one or both homology arms can be shortened to avoid overlap with certain sequence repeat elements, e.g. , Alu repeats, LINE elements, etc.

[1040] 6.3 Quantitative measurements of on-target and off-target gene editing

[1041] It should be noted that the genome editing systems of the present disclosure allow for the detection and quantitative measurement of on-target and off-target gene editing outcomes. The compositions and methods described herein can rely on the use of PCR primer sequences to amplify the genomic locus comprising the expected cut site of the RNA-guided nuclease. In some embodiments, the genomic locus is within a liver-expressed gene. In some embodiments, the liver-expressed gene is LPA. ANGPTL3, PCSK9, I. DLR. APOC2, APOC3, APOB, MTP, ANGPTL4. ANGPTL8. APOA5, ApoB, APOE, IDOL. NPC1L1, ASGRL TM6SF2, GALNT2, LPL, MLXIPL, SORT1. TRIBL MARC1. ABCG5, ABCG8, PNPLA3, TM6SF2, HFE. GCKR HMOX-L UGT1A1, STAP1, LDLRAP1, LMF-1, GP1HBP1, CYP27AI, LIPA or ATP7B. In some embodiments, the primers comprise an adaptor tail for use in a two-step PCR amplification process to prepare amplicon libraries for Next Generation Sequencing (NGS) analysis. A non-limiting example for primers and amplification site used for assessing the on- target genome editing efficiency of an LPA target site set forth in [SEQ ID NO: 24], an ANGPTL3 target site set forth in [SEQ ID NO: 312], and .PCSK9 target site set forth in [SEQ ID NO: 334] are found in Tables 8A-8C. Another non-limiting example for the primers and amplification site used for assessing the on-target genome editing efficiency of an LPA target site set forth in [SEQ ID NO: 26], an ANGPTL3 target site set forth in [SEQ ID NO: 317], and PCSK9 target site set forth in [SEQ ID NO: 335re found in Tables 9A-9C.

[1042] Table 8A: First Exemplary Primer and Amplicon Sequences for an On-Target LPA cut site analysis

[1043] Table 8B: First Exemplary Primer and Amplicon Sequences for an On-Target ANGPTL3 cut site analysis

[1044] Table 8C: First Exemplary Primer and Amplicon Sequences for an On-Target PCSK9 cut site analysis

[1045] Table 9A: Second Exemplary Primer and Amplicon Sequences for an On-Target LPA cut site analysis

[1046] Table 9B: Second Exemplary Primer and Amplicon Sequences for an On-Target

[1047] ANGPTL3 cut site analysis

[1048] Table 9C: Second Exemplary Primer and Amplicon Sequences for an On-Target PCSK9 cut site analysis

[1049] In certain embodiments, the genome editing systems or RNP complexes disclosed herein have minimal or no off-target effects. In certain embodiments, the off-target effect of an RNP is measured by Digenome-seq analysis (Kim et al.. Nature Methods (2015); 12:237-243). In certain embodiments, the off-target effect of a genome editing system or RNP complex is indicated by an off-target count as measured by Digenome-seq analysis. In certain embodiments, the off-target count is measured by Digenome-seq analysis at 1000 nM of the RNP complex. In certain embodiments, the off-target count is measured by the Digenome-seq analysis at 100 nM of the RNP complex.

[1050] In certain embodiments, the off-target count as measured by Digenome-seq analysis of an RNP complex disclosed herein at 1000 nM is less than about 20. less than about 19, less than about 18, less than about 17. less than about 16, less than about 15, less than about 14, less than about 13, less than about 12, less than about 11, less than about 10, less than about 9, less than about 8, less than about 7, less than about 6, less than about 5, less than about 4, less than about 3, less than about 2, or less than about 1. In certain embodiments, the off-target count of an RNP complex disclosed herein as measured by Digenome-seq at 1000 nM is zero or is about zero. In certain embodiments, the off-target count of the RNPs disclosed herein as measured Digenome-seq at 100 nM is less than about 20, less than about 19, less than about 18, less than about 17, less than about 16, less than about 15, less than about 14, less than about 13, less than about 12, less than about 11, less than about 10, less than about 9, less than about 8, less than about 7, less than about 6, less than about 5, less than about 4, less than about 3, less than about 2, or less than about 1. In certain embodiments, the off-target count of an RNP complex disclosed herein as measured by Digenome-seq at 100 nM is zero or is about zero.

[1051] In some embodiments, a quantitative method of assessing the on-target and off-target sites, e.g., GUIDE-Seq, includes the integration of an exogenous double stranded oligo nucleotide (dsODN) tag into the genome. For example, Tsai et al., 2016; Tsai et al., 2014; and Tycko et., 2016, which are incorporated by reference herein in their entirety, describe compositions and methods which allow for the quantitative analysis of off-target and on-target gene editing outcomes, by the integration of a dsODN into RNA guided nuclease (RGN) induced double strand breaks (DSBs). In some embodiments, the dsODN tag is a 34 bp, blunt, 5’ phosphorylated, phosphorothioate linked polynucleotide that becomes incorporated into double stand breaks. The dsODN tag contains a priming site that allows for the amplification, sequencing, and discovery of RNG induced double strand breaks. Non-limiting examples of the dsODN tag and primer are set forth in Table 10.

[1052] Table 10: Exemplary Primer and Amplicon Sequences for an Off-Target cut site analysis

[1053] P represents a 5' phospho rylation and * indicates a phosphorothioate linkage.

[1054] 7. Implementation of genome editing systems: Delivery, Formulations, and Routes of Administration

[1055] As discussed above, the genome editing systems of this disclosure can be implemented in any suitable manner, meaning that the components of such systems, including without limitation the RNA-guided nuclease. gRNA. and optional donor template nucleic acid, can be delivered, formulated, or administered in any suitable form or combination of forms that results in the transduction, transfection, expression or introduction of a genome editing system and / or causes a desired repair outcome in a cell, tissue or subject. The genome editing systems according to this disclosure can incorporate multiple gRNAs, multiple RNA-guided nucleases (or polynucleotides (e.g., mRNA) encoding such RNA-guided nucleases), and other components such as proteins, and a variety of implementations will be evident to the skilled artisan based on the principles illustrated in systems of the disclosure. In some embodiments the genome editing system of the disclosure is delivered into cells as a ribonucleoprotein (RNP) complex. In some embodiments, one or more RNP complexes are delivered to the cell sequentially in any order, or simultaneously. In some embodiments the genome editing system described herein is delivered into cells (e.g., via an LNP) as a gRNA and an RNA encoding an RNA- guided nuclease protein. Tables 11 and 12 set forth several, non-limiting examples of genome editing system implementations. Those of skill in the art will appreciate, however, that these listings are not comprehensive, and that other implementations are possible. With reference to Table 11 in particular, the table lists several exemplary implementations of a genome editing system comprising a single gRNA and an optional donor template. However, genome editing systems according to this disclosure can incorporate multiple gRNAs, multiple RNA-guided nucleases, and other components such as proteins, and a variety of implementations will be evident to the skilled artisan based on the principles illustrated in the table. In the table, [N / A] indicates that the genome editing system does not comprise the indicated component.

[1056] Table 11: Genome Editing System Components

[1057] Table 12 summarizes various delivery methods for the components of genome editing systems, as described herein. Again, the listing is intended to be exemplary rather than limiting.

[1058] Table 12; Summary of delivery methods for components of genome editing systems

[1059] 7.1 Nucleic acid-based delivery of genome editing systems

[1060] Nucleic acids encoding the various elements of a genome editing system according to the present disclosure can be administered to subjects or delivered into cells by art-known methods or as described herein. For example, RNA-guided nuclease-encoding and / or gRNA- encoding DNA, as well as donor template nucleic acids can be delivered by, e.g., vectors (e.g , viral or non-viral vectors), non-vector-based methods (e.g , using naked DNA or DNA complexes), or a combination thereof. In some embodiments the genome editing system of the disclosure are delivered by AAV. Non-limiting examples of AAV serotypes include AAV1, AAV2. AAV3, AAV4, AAV5, AAV6. AAV7, AAV8, AAV9. AAV10, AAV11, AAV12, AAV13. In certain embodiments, the AAV serotypes can be matched to target cell ty pes. For example, in certain non-limiting embodiments, AAV serotypes selected from the group consisting of AAV3, AAV5, AAV8, and AAV9 can be used to target liver cells.

[1061] 7. 1.1 Non-Viral Vectors

[1062] Non-viral vectors can be used to deliver nucleic acids encoding genome editing systems according to the present disclosure. One important category of non-viral nucleic acid vectors are nanoparticles, which can be organic or inorganic. Nanoparticles are well known in the art and are summarized in Cotta-Ramusino. Any suitable nanoparticle design can be used to deliver genome editing system components or nucleic acids encoding such components. For instance, organic (e.g., lipid and / or polymer) nanoparticles can be suitable for use as delivery vehicles in certain embodiments of this disclosure. In one embodiment, the vector is a lipid nanoparticle (LNP).

[1063] In certain embodiments, the LNP can have a diameter of less than 1000 nm, 500 nm, 250 nm, 200 nm, 150 nm, 100 nm, 75 nm, 50 nm, or 25 nm. In some embodiments, the LNP has a size ranging from about 1 nm to about 1000 nm. In some embodiments, the LNP has a size ranging from about 1 nm to about 500 nm. In some embodiments, the LNP has a size ranging from about 1 nm to about 250 nm. In some embodiments, the LNP has a size ranging from about 25 nm to about 200 nm. In some embodiments, the LNP has a size ranging from about 25 nm to about 100 nm. In some embodiments, the LNP has a size ranging from about 35 nm to about 75 nm. In some embodiments, the LNP has a size ranging from about 25 nm to about 60 nm.

[1064] In certain embodiments, but not by way of any limitation, LNPs can be made from ionizable lipids (e.g., cationic lipids), neutral lipids, structural lipids, helper lipids, and PEGylated lipids, or a combination of these. In some embodiments, fusogenic phospholipids (e.g., DOPE) and / or sterols (e.g., cholesterol), may be included in LNPs as 'helper lipids " to enhance transfection activity and / or LNP stability'. Exemplary lipids and polymers for use in nanoparticle formulations, and / or gene transfer are shown in Table 13, and Table 14.

[1065] Table 13: Lipids Used for LNPs and / or Gene Transfer

[1066] Table 14: Polymers Used for LNPs and / or Gene Transfer

[1067] In certain embodiments, the LNPs of the present disclosure comprise a cationic lipid, e.g., those cationic lipids disclosed in Table 13. In certain embodiments, the cationic lipids that find use in the compositions and methods of the present disclosure include, but are not limited to: l,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA); 1 ,2-dilinolenyloxyN, N- dimethylaminopropane (DLenDMA); 2.2-dilinoleyl-4-(2-dimethyl-aminoethyl)-

[1068] [1.3]dioxolane (DLin-K-C2-DMA.; '"XTC2"'); 2,2-dilinoleyl-4-(3-dimethyl-aminoopropyl)-

[1069] [1.3]-di oxolane (DLin-K-C3-DMA); 2, 2-dilinoieyl-4-(4-dimethylaminobutyl)-ll,3]-di oxolane (DLinK-C4-DMA); 2,2-dilinoleyl-5-dimethylaminomethyl-[J ,3]-dioxane (DLin-K6-DMA);

[1070] 2.2-dilinoleyl-4-N-methylpepiazino-[l,3]-dioxolane (DLin-K-MPZ), 2,2-dilinoleyl-4- dimethylaminomethyl-[l,3]-dioxolane (DLin-K-DMA); 1 ,2-dilinoleylcarbamoyl-oxy-3- dim. ethylaminopropane (DLin-C-DAP); l,2-dilinoleyoxy-3-(dimethylamino) acetoxypropane (DLin-MAC); l,2-dilinoleyoxy-3-morpholinopropane (DLin-MA); L2-dilinoleoyl-3- dimethylaminopropane (DLinDAP), l,2-dilinoleylthio-3-dimethylaminopropane (DLin- SDMA); l-linoleoyi-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP); 1,2- dilinoleyloxy-3-trirmethylaminopropane chloride salt (DLin-TMA Cl), 1 ,2-dilinoleoyl-3- trimethylaminopropane chloride salt (DLin-TAP Cl); l,2-dilinoleyloxy-3-(N- methylpiperazino)propane (DLin-MPZ); 3-(N,N-dilinoleylamino)-l,2-propanediol (DLinAP); 3-(N,N-di oleylamino)-! ,2-propanedio (DOAP); l,2-dilinoleyloxo-3-(2-N,Ndimethylamino) ethoxypropane (DLin-EG-DMA); N,N-dioleyl-N.N-dimethylammonium chloride (DODAC);

[1071] 1.2-dioleyloxy-N,N-dimethylanrinopropane (DODMA); 1 ,2-distearyloxyN,N-dimethylamino- propane (DSDMA); N-(l-(2,3-dioley loxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA); N,N-distearyl-N,N-dimethylammonium bromide (DDAB); N-(l-(2,3-dioleoyloxy) propyl)-N,N,N -trimethylammonium chloride (DOTAP); 3-(N - (N',N'-dimethylamino- ethane)-carbanloyl)cholesterol (DC-Chol); N-(l,2-dim.yristyloxyprop-3-yl)-N,N-dimethyl-N- hydroxyethyl ammonium bromide (DMRIE); 2,3- dioleyioxy-N-[2(spermine- carboxmnido)ethyl] -N,N-dimethyl-l-propanaminiumtrifluoroacetate (DOSPA); dioctadecylamidoglycyl spermine (DOGS); 3-dimethylamino-2-(cholest-5-en-3-betaoxybutan-4-oxy)-l- (cis,cis-9, 12-octadecadienoxy)propane (CLinDMA); 2-[ 5'-(cholest-5-en-3-beta-oxy)-3'- oxapentoxy)-3-dimethy l-l-(cis,cis-9', 1 -2'-octadecadienoxy) propane (CpLinDMA); 1,2- Dilinoleoy Icarbamy 1-3-dimethyiaminopropane (DLinCDAP); N-dimethyi-3,4- dioleyloxybenzylamine (DMOBA); and 1.2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP).

[1072] In certain embodiments, the cationic lipids that find use in the compositions and methods of the present disclosure include, but are not limited to: (9Z,12Z)-3-((4,4- bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9, 12-dienoate (also known as LP-01) or a pharmaceutically acceptable salt thereof. In certain embodiments, the cationic lipids that find use in the compositions and methods of the present disclosure include, but are not limited to: (9Z,9'Z,12Z,12'Z)-2-(((3-(4- methylpiperazin-l-yl)propanoyl)oxy)methyl)propane-1.3-diyl bis(octadeca-9,12-di enoate);

[1073] (9Z,9'Z,12Z.12'Z)-2-(((4-(pynolidin-l-yl)butanoyl)oxy)methyl)propane-1.3- diy lbis(octadeca-9, 12- di enoate); (9Z,9'Z, 12Z, 12'Z)-2-(((4-(piperidin- 1 - yl)butanoyl)oxy)methyl)propane-l,3-diyl bis(octadeca-9,12-dienoate); (9Z,9Z,12Z,2'Z)-2- (((l,4-dimethylpiperidine-4-carbonyl)oxy)methyl)propane-l,3-diyl bis(octadeca-9,12- dienoate); (9Z,9'Z,12Z,12'Z)-2-(((l-(cyclopropylmethyl)piperidine-4- carbonyl)oxy)methyl)propane-l,3-diyl bis(octadeca-9,12-dienoate); (9Z,12Z)-3-((4,4- bis(octyloxy)butanoyl)oxy)-2-((((3-(dimethylamino)propoxy)carbonyl)oxy)methyl) propyl octadeca-9.12-di enoate; (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((((l-ethylpiperidin- 3-yl)methoxy)carbonyl)oxy)methyl)propyl octadeca-9, 12-dienoate; 2-((((2- (diethylamino)ethoxy)carbonyl)oxy)methyl)propane-l,3-diylbis(2-heptylundecanoate); (9Z,12Z)-3-(((2-(diethylamino)ethoxy)carbonyl)oxy)-2-(((2-heptylundecanoyl)oxy) methy l)propy 1 octadeca-9, 12-dienoate; 2-((((3 -(dimethylamino)propoxy)carbony 1) oxy)methyl)propane-l,3-diyl bis(2 -heptylundecanoate); (9Z,12Z)-3-(((3-

[1074] (diethylamino)propoxy)carbonyl)oxy)-2-(((2-heptylundecanoyl)oxy)methyl)propyl octadeca- 9,12-dienoate; (9Z,12Z)-3-(((2-(dimethylamino)ethoxy)carbonyl)oxy)-2-(((3- octylundecanoyl)oxy)methyl)propyl octadeca-9, 12-dienoate; 2-((((3-

[1075] (diethy lamino)propoxy)carbonyl)oxy)methyl)propane- 1 ,3 -diyl bis(3-octylundecanoate); (9Z,12Z)-3-(((3-(diethylamino)propoxy)carbonyl)oxy)-2-(((3-octylundecanoyl)oxy)methyl )propyl octadeca-9, 12-dienoate; (9Z, 12Z)-3-(((3-(diethylamino)propoxy)carbonyl)oxy)-2- (((7-hexyltridecaonoyl)oxy)methy l)propyl octadeca-9, 12-di enoate; (9Z, 12Z)-3-(((3-

[1076] (diethylamino)propoxy)carbonyl)oxy)-2-(((9-pentyltetradecanoyl)oxy)methyl)propyl octadeca-9, 12-dienoate; (9Z,12Z)-3-(((3-(diethylamino)propoxy)cabonyl)oxy)-2-(((5- heptyldodecanoyl)oxy)methyl)propyl octadeca-9, 12-di enoate; (9,12Z)-3-(2,2- bis(heptyloxy)acetoxy)-2-((((2-(dimethylarnino)ethoxy)carbonyl)oxy)methyl)propyl octadeca-9.12-di enoate; (9Z,12Z)-3-((6,6-bis(octyloxy)hexanoyl)oxy)-2-((((3-

[1077] (diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9.12-dienoate; 2-(3-ethyl-l 1- (((9Z, 12Z)-octadeca-9, 12-di enoyl oxy )methyl)-8,14-dioxo-7, 9, 13-trioxa-3-azaheptad ecan-17- yl)propane- 1,3 -diyl dioctanoate; (9Z,9'Z,12Z,12Z)-2-((((3-

[1078] (dimethylamino)propoxy)carbonyl)oxy)methyl)propane-l,3-diyl bis(octadeca-9, 12-dienoate); (9Z,9'Z,12Z,12'Z)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propane-l,3-diyl bis(octadeca-9, 12-dienoate); (9Z,9'Z,12Z,12'Z)-2-((((2-

[1079] (dimethylamino)ethoxy)carbonyl)oxy)methyl)propane-l,3-diyl bis(octadeca-9, 12-dienoate);

[1080] 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((9Z, 12Z)-octadeca-9, 12-di enoyloxy )methyl)propyl 1- isopropylpiperidine-4-carboxylate; 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((9Z,12Z)- octadeca-9.12-dienoyloxy)methyl)propyl l-(cyclopropylmethyl)piperidine-4-carboxylate; 3- ((4,4-bis(octyl oxy )butanoy l)oxy)-2-(((9Z, 12Z)-octadeca-9, 12-dienoyloxy )methy l)propyl 1 - methylpyrrolidine-3-carboxylate; (9Z,9'Z,12Z,12Z)-2-((((3-

[1081] (diethylamino)propoxy)carbonyl)oxy)methyl)-2-(((9Z, 12Z)-octadeca-9, 12- di enoyloxy )methyl)propane- 1.3 -diyl bis(octadeca-9.12-dienoate); (2S)-3-((4,4- bis(octyloxy)butanoyl)oxy)-2-(((9Z,12Z)-octadeca-9,12-dienoyloxy)methyl)propyl 1- methylpyrrolidine-2-carboxylate; (2R)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((9Z,12Z)- octadeca-9.12-dienoyloxy)methyl)propyl l-methylpyrrolidine-2-carboxylate; 3-((4,4- bis(octyloxy)butanoyl)oxy)-2-(((9Z,12Z)-octadeca-9,12-dienoyloxy)methyl)propyl 4- methylmorpholine-2-carboxylate; 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((9Z, 12Z)-octadeca- 9, 12-di enoyl oxy)methyl)propyl l,4-dimethylpiperidine-4-carboxylate; 3-((4,4- bis(octyloxy)butanoyl)oxy)-2-(((9Z,12Z)-octadeca-9,12-dienoyloxy)methyl)propyl 1- methylpiperidine-4-carboxylate; (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((((l- methylpyrrolidin-3-yl)oxy)carbonyl)oxy)methyl)propyl octadeca-9, 12-dienoate; (9Z, 12Z)-3- ((4,4-bis(octyloxy)butanoyl)oxy)-2-(((((l-methylpiperidin-4- yl)oxy)carbonyl)oxy)methyl)propyl octadeca-9, 12-dienoate; (9Z,12Z)-3-((4,4- bis(octyloxy)butanoyl)oxy)-2-(((((l-methylazetidin-3-yl)oxy)carbonyl)oxy)methyl)propyl octadeca-9.12-dienoate; (9Z,12Z)-3-((4.4-bis(octyloxy)butanoyl)oxy)-2-(((((l-ethylpiperidin-

[1082] 4-yl)oxy)carbonyl)oxy)methyl)propyl octadeca-9, 12-dienoate; (9Z,12Z)-3-((4,4- bis(octyloxy)butanoyl)oxy)-2-(((((l-methylpiperidin-4- yl)methoxy)carbonyl)oxy)methyl)propyl octadeca-9, 12-di enoate; (9Z,12Z)-3-((4,4- bis(octyloxy)butanoyl)oxy)-2-(((((l,2,2,6,6-pentamethylpiperidin-4- yl)oxy)carbonyl)oxy)methyl)propyl octadeca-9, 12-di enoate; (9Z, 12Z)-3-((4,4- bis(octyloxy)butanoyl)oxy)-2-((((3-(dimethylamino)propyl)carbamoyl)oxy)methyl)propyl octadeca-9.12-di enoate; 3-((4.4-bis((2-propylpentyl)oxy)butanoyl)oxy)-2-(((9Z,12Z)- octadeca-9.12-dienoyloxy)methyl)propyl 1 ,4-dimethylpiperidine-4-carboxylate; 3-((6,6- bis((2-propylpentyl)oxy )hexanoyl)oxy)-2-(((9Z,12Z)-octadeca-9, 12- di enoyloxy)methyl)propyl l,4-dimethylpiperidine-4-carboxylate; 2-(((l-methylpyrrolidine-3- carbonyl)oxy)methyl)propane- 1,3 -diyl bis(4,4-bis(octyloxy)butanoate); 2-(((l- methylpyrrolidine-3-carbonyl)oxy)methyl)propane-1.3-diyl bis(6,6-bis(octyloxy)hexanoate); 2-(((l-methylpyrrolidine-3-carbonyl)oxy)methyl)propane-l,3-diyl bis(6,6-bis((2- propylpentyl)oxy)hexanoate); 2-(5-(3-(dodecanoyloxy)-2-(((l-methylpyrrolidine-3- carbonyl)oxy)methyl)propoxy)-5-oxopentyl)propane-l,3-diyl dioctanoate; 2-(5-(3-((l- methylpyrrolidine-3-carbonyl)oxy)-2-((palmitoyloxy)methyl)propoxy)-5-oxopentyl)propane- 1 ,3 -diyl dioctanoate; 2-(S-(3-(( l-methylpyrrolidine-3-carbonyl)oxy)-2-

[1083] ((tetradecanoyloxy)methyl)propoxy)-S-oxopentyl)propane-l,3-diyl dioctanoate; 3-((4,4- bis(octyl oxy)butanoyl)oxy)-2-((dodecanoyloxy)methyl)propyl 1 -methylpyrrolidine-3- carboxylate; 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((tetradecanoyloxy)methyl)propyl 1 - methylpyrrolidine-3-3-((4.4-bis(octyloxy)butanoyl)oxy)-2-((palmitoyloxy)methyl)propyl 1- methylpyrrolidine-3-carboxylate; 1 -(3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((l - methylpyrrolidine-3-carbonyl)oxy)methyl)propyl) 8-methyl octanedioate; l-(3-((4,4- bis(octyloxy)butanoyl)oxy)-2-(((l-methylpyrrolidine-3-carbonyl)oxy)methyl)propyl) 10- octyl decanedioate; l-(3-((6,6-bis((2-propylpentyl)oxy)hexanoyl)oxy)-2-(((l,4- dimethylpiperidine-4 carbonyl)oxy)methyl)propyl) 10-octyl decanedioate; l-(3-((6,6-bis((2- propylpentyl)oxy)hexanoyl)oxy)-2-(((l,4-dimethylpiperidine-4-carbonyl)oxy)methyl)propyl) 8-methyl octanedioate; and 8-dimethyl O'l,Ol-(2-(((l-methylpyrrolidine-3- carbonyl)oxy)methyl)propane- 1.3 -diyl) dioctanedioate.

[1084] In certain embodiments, the cationic lipids that find use in the compositions and methods of the present disclosure include, but are not limited to: ((4,4’-((((3- (Dimethylamino)propyl)thio)carbonyl) azanediyl)bis(butanoyl))bis(oxy))bis(propane-2, 1,3- triyl) tetranonanoate; ((4,4'-((((3-(Dimethylamino)propyl)thio)carbonyl) azanediyl)bis (butanoyl))bis(oxy)) bis(propane-2,l,3-triyl) tetraoctanoate; bis(l,3-bis(Nonanoyloxy)propan- 2-yl) 5-((4-(dimethylamino)butanoyl)oxy)nonanedioate HCI salt; bis(l,3-bis(Octanoyloxy) propan-2-yl) 5-((4-(dimethylamino) butanoyl)thio)nonanedioate; bis(l,3-bis(N onanoyloxy) propan-2-yl) 4-((4-(dimethylamino) b utanoyl)oxy)heptanedioate; bis(l,3-bis(Octanoyloxy) propan-2-yl) 4-((4-(dimethylamino) butanoyl)thio)heptanedioate; ((2,2'-((((3-(Dimethyl- amino)propyl)thio) carbonyl)azanediyl)bis(acetyl))bis(oxy))bis(propane-2, 1 ,3 -triyl) tetranonanoate; bis(l,3-bis(N onanoyloxy )propan-2-yl) 4-((4-(dimethylamino) butanoyl)thio) heptanedioate; ((4,4'-((((3-(Dimethylamino)propyl)thio)carbonyl) azanediyl)bis (butanoyl)) bis(oxy))bis(propane-2,l,3-triyl) tetrakis(3-cyclohexylpropanoate); ((4.4'-((((3-(Dimethyl- amino)propyl)thio)carbonyl) azanediyl)bis(butanoyl))bis(oxy)) bis(propane-2,l ,3-triyl) tetrakis(4-cyclohexyl butanoate); ((6,6'-((((3-(Dimethylamino)propyl)thio)carbonyl) azanedi- yl)bis(hexanoyl))bis(oxy))bis(propane-2,l,3-triyl)tetrakis(3-cyclohexylpropanoate);

[1085] Nonanoic acid 2-(3-{(3-dimethylaminopropylsulfanylcarbonyl)-[2-(2-nonanoyloxy-l- nonanoyloxymethyl-ethoxycarbonyl)-ethyl]amino} -propionyloxy )-3-octanoyloxy -propyl ester; ((4,4'-((((3-(Dimethylamino)propyl)thio)carbonyl) azanediyl)bis(butanoyl))bis(oxy))bis (propane-2, 1,3 -triyl) tetrakis(2-(4-methylcyclohexyl) acetate); ((4,4 '-((((3-

[1086] (Dimethylamino)propyl)thio)carbonyl) azanediyl)bis(butanoyl))bis(oxy)) bis(propane-2, 1,3- triyl) tetrakis(4-ethylcyclo hexane- 1 -carboxylate); ((4,4'-((((3-(Dimethylamino) propyl)thio)carbonyl) azanediyl)bis(butanoyl)) bis(oxy))bis(propane-2,l,3-triyl) tetrakis(3- cyclohexyl-2-methylpropanoate); ((4,4'-((((3-(Dimethylamino)propyl)thio)carbonyl) azanediyl)bis(butanoyl)) bis(oxy))bis(propane-2,l,3-triyl) tetrakis(2 -methyloctanoate); ((4.4 '- ((((3-(Dimethylamino)propyl)thio)carbonyl) azanediyl) bis(butanoyl)) bis(oxy))bis(propane- 2,l,3-triyl)tetrakis(2,2-dimethylheptanoate); ((3,3'-((((3-(Dimethylamino) propyl)thio)carbonyl) azanediyl) bis(propanoyl)) bis(oxy))bis (methylene))bis(2- methylpropane-2, 1,3 -triyl) tetrakis(3-(4-methyl cyclohexyl)propanoate); ((3,3'-((((3- (Dimethylamino)propyl)thio)carbonyl) azanediyl) bis(propanoyl))bis(oxy)) bis (methylene)) bis(2 -methyl propane-2, 1,3-triyl) tetrakis(2-(4-ethylcyclohexyl) acetate); ((3,3'-((((3- (Dimethylamino)propyl)thio)carbonyl) azanediyl) bis(propanoyl))bis(oxy)) bis(methylene)) bis(2-methyl propane-2, 1,3-triyl) tetrakis(2-(4-ethylcyclohexyl)acetate); ((3,3'-((((3- (Dimethylamino)propyl)thio)carbonyl)azanediyl)bis(propanoyl))bis(oxy))bis(methylene))bis (2-methylpropane-2,l,3-triyl)tetrakis(3,3-dimethylheptanoate); ((3,3'-((((3-(Dimethylamino) propyl)thio)carbonyl)azanediyl)bis(propanoyl))bis(oxy))bis(methyl-ene))bis(2-methyl- propane-2, 1,3-triyl) tetrakis(octanoate); ((4,4'-((((3-(Dimethylamino) propyl)thio)carbonyl) azanediyl) bis(butanoyl))bis(oxy)) bis(methylene))bis(propane-2, 1,3-triyl) tetranonanoate; ((3.3'-((((2-(Dimethylamino)ethyl)thio)carbonyl)azanediyl)bis(propanoyl))bis(oxy)) bis(methylene))bis(propane-2,l,3-triyl)tetranonanoate; ((3,3'-((((3-(Dimethylamino)propyl) thio)carbonyl) azanediyl)bis(propanoyl))bis(oxy))bis(methylene))bis (propane-2, 1,3 -triyl) tetrakis(3-(4-methylcyclohexyl)propanoate); ((4.4'-((((3-(Dimethyl-amino)propyl)thio) carbonyl)azanediyl) bis(butanoyl))bis (oxy)bis(propane-2,l,3-triyl) tetrakis(octahydro-lH- indene); ((4,4 '-((((3-(dimethylamino)propyl)thio)carbonyl)azanediyl) bis(butanoyl))bis(oxy) ) bis(propane-2,l,3-triyl)tetrakis(octahydro-lH-indene-5-carboxylate); ((4,4'-(((3-(dimethyl- amino)propoxy)carbonyl) azanediyl)bis(butanoyl))bis(oxy)) bis(propane-2.1.3 -triyl) tetranonanoate; ((4,4'-(((3-(dimethylamino)propyl)carbamoyl) azanediyl)bis (butanoyl)) bis(oxy))bis(propane-2,l ,3-triyl)tetranonanoate; ((4,4'-((((3-(dimethylamino) propyl)thio) carbonyl)azanediyl)bis(butanoyl)) bis(oxy)) bis(propane-2,l,3-triyl) tetrakis(2-(p- tolyl)acetate); [2-[4-[3-(dimethylamino)propoxycarbonyl-[4-[2-(2-methyloctanoyloxy)-l-(2- methyloctanoyloxymethyl)ethoxy]-4-oxobutyl] amino]butanoyloxy]-3-(2-methyloctanoyl- oxy)propyl] 2-methyloctanoate; [2-[4-[3-(dimethylamino)propylcarbamoyl-[4-[2-(2- methyloctanoyl oxy)-l-(2-methyloctanoyloxymethyl)ethoxy ]-4-oxobutyl ] amino ]butanoyl- oxy]-3-(2-methyloctanoyloxy)propyl] 2-methyloctanoate; [2-[4-[[3-(dimethylamino)propyl- methyl-carbamoyl] [4-[2-(2-methy loctanoyloxy)- 1 -(2-methyloctanoy loxymethyl)ethoxy] -4- oxobutyl] amino] butanoyloxy]-3-(2-methyloctanoyloxy)propyl] 2-methyloctanoate; [2-[4-[5- (dimethylamino)pentanoyl-[4-[2-(2-methyloctanoyloxy)-l-(2-methyloctanoyloxym ethyl)ethoxy]-4-oxo-butyl] amino] butanoyloxy]-3-(2-methyloctanoyloxy)propyl] 2- methyloctanoate; [2-[4-[5-(dimethylamino)pentyl-[4-[2-(2 -methyl octanoyloxy)-l-(2-methyl- octanoyloxym ethyl)ethoxy]-4-oxo-butyl] amino] butanoyloxy] -3 -(2 -methyloctanoy 1- oxy)propyl] 2-methyloctanoate; bis [2-(2-methyloctanoyloxy)-l-(2-methyloctanoyl oxym ethyl)ethyl] 5-[ 4-(dimethylamino)butanoyloxy] nonanedioate; and ((4,4 '-((((3- (dimethylamino) propyl)thio)carbonyl)azanediyl)bis(butanoyl)) bis(oxy)) bis(propane-2, 1,3- triyl) tetrakis(2-methyl-4-(p-tolyl) butanoate).

[1087] In certain embodiments, the cationic lipids that find use in the compositions and methods of the present disclosure include, but are not limited to: 6,6'- (Methylazanediyl)Bis(N,N-Dioctylhexanamide); 6,6'-(Octylazanediyl)Bis(N,N-Dioctylhexan- amide); 6.6'-(Hexylazanediyl)Bis(N,N-Dioctylhexanamide); 10, 10'-(Methylazanediyl)Bis (N.N-Dioctyldecanamide); 8,8'-(Methylazanediyl)Bis(N,N-Didecyloctanamide); 6,6'- (Methylazanediyl)Bis(N,N-Didecylhexanamide); 6,6'-(Methylazanediyl)Bis(N,N-Didodecyl- hexanamide); 6,6'-((2-Hydroxyethyl)Azanediyl)Bis(N,N-Dioctylhexanamide); 6,6'-((6- Hydroxyhexyl)Azanediyl)Bis(N,N-Dioctylhexanamide); 6,6'-((2-Hydroxyethyl)Azanediyl) Bis(N,N-Didecylhexanamide); 8,8'-((6-Hydroxyhexyl)Azanediyl)Bis(N,N-Dioctyloct- anamide); 10,10'-((6-Hydroxyhexyl)Azanediyl)Bis(N,N-Dioctyldecanamide); 10, 10'-((2- Hydroxy ethyl)Azanediyl)Bis(N,N-Didecyldecanamide); 8,8'-((5-Hydroxypentyl)Azanediyl) Bis(N,N-Didecyloctanamide); 8,8'-((4-Hydroxybutyl)Azanediyl)Bis(N,N-Didecyloct- anamide); 8,8'-((6-Hydroxyhexyl)Azanediyl)Bis(N,N-Didecyloctanamide); 8,8'-((2-Hydroxy- ethyl)Azanediyl)Bis(N,N-Didecyloctanamide); 10,10'-((4-Hydroxy butyl) Azanediy l)Bis(N,N- Didecyldecanamide); N,N'-( (Methylazanediyl )Bis(Hexane-6, 1-Diyl) )Bis(N,2- Dihexyldecanamide); N,N'-((Methylazanediyl)Bis(Hexane-6, l-Diyl))Bis(N-Hexyl Palmitamide); (9z,9'z,12z,12'z)-N,N'-((Methylazanediyl)Bis(Hexane-6.L-Diyl))Bis(N- Hexyloctadeca-9,L2-Dienamide); N,N-Didecyl-8-((8-(Hexadecylamino)-8-Oxooctyl) (Methyl)Amino)Octanamide; 8,8'-( (8-(Decylamino )-8-Oxooctyl )Azanediyl )Bis(N,N- Didecyloctanamide); 8,8'-((6-(Dihexylamino)-6-Oxohexyl)Azanediyl)Bis(N,N-Didecyloctan- amide): 8.8'-((5-(Decylamino)-5-Oxopentyl)Azanediyl)Bis(N.N-Didecyloctanamide): 6.6' -( (8-(Decylamino )-8-Oxooctyl )Azanediyl )Bis(N,N-Didecylhexanamide); 6,6'-((2- (Dihexylamino)Ethyl)Azanediyl)Bis(N,N-Didecylhexanamide); 10,10'-((2-(Dimethyl- amino)Ethyl)Azanediyl)Bis(N,N-Didecyldecanamide); 2-Butyloctyl 6-(Bis(6- (Dioctylamino)-6-Oxohexyl)Amino)Hexanamide; 6,6'-((4-Hydroxybutyl)Azanediyl)Bis(N,N- Bis(2-Ethylhexyl)Hexanamide); 8,8'-((2-Hydroxyethyl)Azanediyl)Bis(N,N-Didodecyl- octanamide); 6,6'-((6-Hydroxyhexyl)Azanediyl)Bis(N,N-Didodecylhexanamide); N,N- Didecyl-8-((8-(Hexadecyl(Methyl)Amino)-8-Oxooctyl)(Methyl )Amino)Octanamide; 8,8'- (Methylazanediyl)Bis(N,N-Didodecyloctanamide); 8,8'-((3-Hydroxypropyl)Azanediyl) Bis(N,N-Didecyloctanamide); 8.8'-( (2-(2-Hydroxyethoxy)Ethyl )Azanediyl )Bis(N,N- Didecyloctanamide); 8,8'-((5-Hydroxy-4,4-Dimethylpentyl)Azanediyl)Bis(N,N-Didecyl- octanamide); 8,8'-((3-(2-Methyl-Lh-Imidazol-L-Yl)Propyl)Azanediyl)Bis(N,N-Didecyloctan- amide); 8,8'-((7-Hydroxyheptyl)Azanediyl)Bis(N,N-Didecyloctanamide); 8,8’-( (2-(2-

[1088] Methoxyethoxy)Ethyl)Azanediyl)Bis(N,N-Didecyloctanamide); 8,8'-((8-Hydroxyoctyl) Azanediyl)Bis(N,N-Didecyloctanamide); 8,8'-((3-(Lh-Imidazol-L-Yl)Propyl)Azanediyl) Bis(N,N-Didecyloctanamide); 8,8'-((2,2-Difluoro-3-Hydroxypropyl) Azanediyl)Bis(N,N- Didecyloctanamide); 8,8'-((3-((2-(Methylamino)-3,4-Dioxocyclobut-L-En-Lyl) Amino) Propyl)Azanediyl)Bis(N,N-Didecyloctanamide); 8,8'-((2-Fluoro-3-Hydroxypropyl)

[1089] Azanediyl)Bis(N,N-Didecyloctanamide); 8,8'-((3,3,3-Trifluoro-2-(Hydroxymethyl)Propyl) Azanediyl) Bis(N,Ndidecyloctanamide); 8,8'-((5-Methoxypentyl)Azanediyl)Bis(N,N- Didecyloctanamide); N,N-Didecyl-8-((8-(Dioctylamino)-8-Oxooctyl)(Methyl)Amino) Octanamide; Tert-Butyl (3-(Bis(10-(Didecylamino)-10-oxodecyl)Amino)Propyl)Carbamate; 10,10'-((3-(Lh-Imidazol-L-Yl)Propyl)Azanediyl)Bis(N.N-Didecyldecanamide); 8,8'- (Methylazanediyl)Bis(N,N-Dinonyloctanamide); Tert-Butyl (3-(Bis( 10-(Didecylamino )-10- oxodecyl )Amino )Propyl ) Carbamate; 10,10'-((3-((2-(Methylamino)-3,4-Dioxocyclobut-L- En-Lyl)Amino)Propyl) Azanediyl) Bis(N,N-Didecyldecanamide); N.N'-((methylazanediyl) bis(octane-8, 1 -diyl))bis(N-hexylhexanamide); N.N'-(((5-hydroxypentyl)azanediyl)bis (octane-8, l-diyl))bis(N-hexylhexanamide); N.N'-((methylazanediyl)bis(octane-8, 1- diyl))bis(N-octyloctanamide); N.N'-(((5-hydroxypentyl)azanediyl )bis(octane-8, 1 -diyl))bis(N- octyloctanamide); N,N'-((methylazanediyl)bis(octane-8, l-diyl))bis(N-octyloctanamide); N,N'-(((5-hydroxypentyl)azanediy l)bis(octane-8, 1 -diy l))bis(N-decy Idecanamide); N,N'- ((methylazanediyl)bis(octane-8, 1 -diyl))bis(N-dodecyldodecanamide); N,N'-(((5- hydroxypentyl)azanediyl)bis( octane-8, l-diyl))bis(Ndodecyldodecanamide); N,N'- ((methylazanediyl)bis(octane-8, l-diyl))bis(2-hexyldecanamide); N,N'-((methylazanediyl)bis (octane-8, l-diyl))bis(2-hexyl-N-methyldecanamide); N.N'-(((5-hydroxypentyl)azanediyl)bis (octane-8, l-diyl))bis(2-hexyldecanamide); N,N'-(((5-hydroxypentyl)azanediyl)bis( octane-8, l-diyl))bis(2-hexyl-N methyldecanamide); N-decyl-N-(8-((8-(didecylamino)-8 oxooctyl) (methyl) amino) octyl)decanamide; N-decyl-N-(8-((8-(didecylamino)-8-oxooctyl)(5- hydroxypentyl)amino)octyl)decanamide; 6,6'-((5-hydroxypentyl)azanediyl)bis(N.N-didecyl- hexanamide); 7, 7'-(methylazanediyl)bis(N.N-didecylheptanamide); 8.8'-((2-(dimethylamino) ethyl)azanediyl)bis(N,N-didecyloctanamide); 8,8'-((2-(pyrrolidin-l-yl)ethyl)azanediyl) bis(N,N-didecyloctanamide); N,N-didecyl-8-((8-(decyl(methyl)amino)-8-oxooctyl)(methyl) amino)octanamide; N,N-didecyl-8-((8-(decyl(methyl)amino)-8-oxooctyl)(2-hydroxyethyl) amino)octanamide; 8,8'-((5-Hydroxypentyl)Azanediyl)Bis(N.N-Dinonyloctanamide); 8,8'- ((5-Hydroxypentyl)Azanediyl)Bis(N,N-Didecyl-2-Fluorooctanamide); 8,8'-(Methyl- azanediyl)Bis(N,N-Didecyl-2-Fluorooctanamide); 2,2'-((5-Hydroxypentyl)Azanediyl) Bis(N,N-Didecylacetamide); and 4,4'-((5-Hydroxypentyl)Azanediyl)Bis(N,N-Didecylbutan- amide).

[1090] Moreover, each of the following references, which disclose ionizable cationic lipids and other lipid components that find use in connection with the compositions and methods disclosed herein, is hereby incorporated by reference in its entirety7: international patent application publications W02015 / 095340, W02022 / 011156, WO2022 / 133344,

[1091] WO2023 / 086514, WO2023 / 081776, W02023 / 010128. WO2022 / 235972. WO2022 / 235935, WO2022 / 235923, WO2022 / 056413, W02022 / 036170, W02020 / 191103, WO2018 / 183901, WO2018 / 119163, W02015074085A1, W02016081029A1, WO2017117530A1,

[1092] W02019191780A, W02020 / 097548, W02020 / 097540, W02020 / 097520, W02020 / 097493, WO2016 / 197133, WO2011 / 141705, WO2011 / 141704. WO2011 / 000107, WO2011 / 000106, WO2010 / 144740, W02010 / 129709,W02010 / 088537, WO2010 / 054406, WO2010 / 054405, W02010 / 054401, W02010 / 054384, W02009 / 127060, W02008 / 042973, W02007 / 012191, W02006 / 074546, WO2005 / 121348, W02005 / 120461. W02005 / 120152, W02005 / 026372, W02005 / 007196, W02004 / 002453, W02002 / 087541, W02000 / 003683, WO2023 / 114944, WO2023 / 114943, WO2023 / 114937, W02022 / 016070, WO2021 / 030701, W02020 / 146805, W02020 / 081938, W02020 / 061426, WO2019 / 089828, WO2019 / 036030, W02019 / 036028, W02019 / 036008, WO2019 / 036000, WO2018 / 200943. WO2018 / 191719, WO2018 / 191657, WO2018 / 081480, WO2018 / 078053, WO2017 / 117528. WO2017 / 075531. WO2017 / 004143, WO2015 / 199952; W02013 / 12680; US patent numbers US10227302, US10383952, and US10526284; and US application publication US2020 / 0297634.

[1093] In certain embodiments, the LNPs of the present disclosure comprise a non-cationic lipid, e.g., those non-cationic lipids disclosed in Table 13. In certain embodiments, the noncationic lipids that find use in the compositions and methods of the present disclosure include, but are not limited to lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC). dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl phosphatidyl ethanol amine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleyol-phosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N- maleimidomethyl)-cyclohexane-lcarboxylate (DOPE-mal), dipalmitoyl-phosphatidyl- ethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), monomethyl-phosphatidyl ethanol amine, phosphatidylethanolamine (DEPE), dimethyl-phosphatidylethanolamine. dielaidoylstearoyloleoylphosphatidylethanolamine (SOPE).

[1094] In certain embodiments, the PEGylated lipids comprise a PEG molecule with a molecular weight from about 200Dato about 5000Da. In certain embodiments, the PEGylated lipids comprise a PEG molecule with a molecular weight of 2000Da (2kDa).

[1095] In certain embodiments, the PEG-lipids can include, but are not limited to, those identified in Table 14. For example, but not by way of limitation, The PEG-lipid can comprise PEG coupled to dialkyloxypropyls (PEG-DAA), PEG coupled to diacylglycerol (PEG-DAG), methoxypoly ethylenegly col (PEG-DMG or PEG2000-DMG), PEG coupled to phospholipids such as phosphatidylethanolamine (PEG-PE), PEG conjugated to ceramides, PEG conjugated to cholesterol or a derivative thereof, and mixtures thereof. In certain embodiments, the PEG- lipid comprises detergent-like PEG lipids (e.g., PEG-DSPE).

[1096] In certain embodiments, the PEG moiety is conjugated directly to the lipid. In certain embodiments, the PEG moiety is conjugated to the lipid via a linker moiety. Any linker moiety suitable for conjugating the PEG to a lipid can be used including, but not limited to, ester- containing linker moi eties and / or non-ester-containing linker moieties. In certain embodiments, an ester-containing linker moiety is used to conjugate the PEG to the lipid. Exemplary ester-containing linker moieties include, e.g., carbonate (-OC(O)O-), succinoyl, phosphate esters (-O-(O)POH-O-), sulfonate esters, and combinations thereof. Exemplary' nonester containing linker moieties include, but are not limited to, amido (-C(O)NH-), amino (- NR-), carbonyl (-C(O)-), carbamate (-NHC(O)O-), urea (-NHC(O)NH-), disulfide (-S-S-), ether (-0-), succinyl (-(O)CCH2CH2C(O)-), succinamidyl (-NHC(O)CH2CH2C(O)NH-), ether, as well as combinations thereof (such as a linker containing both a carbamate linker moiety and an amido linker moiety).

[1097] In certain embodiments, phosphatidylethanolamines having a variety of acyl chain groups of varying chain lengths and degrees of saturation can be conjugated to PEG to form the PEG-lipid conjugate. In certain embodiments, phosphatidylethanolamines comprising saturated or unsaturated fatty' acids with carbon chain lengths in the range of CIO to C20 are employed in connection with the compositions and methods disclosed herein. In certain embodiments, phosphatidylethanolamines with mono- or di-unsaturated fatty acids and mixtures of saturated and unsaturated fatty' acids can also be used. In certain embodiments, the phosphatidylethanolamines that find use in connection with the compositions and methods disclosed herein include, but are not limited to, dimyristoyl- phosphatidylethanolamine (DMPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dioleoylphosphatidylethanolamine (DOPE), and distearoyl-phosphatidylethanolamine (DSPE).

[1098] In certain embodiments the PEG-DAA conjugate of the instant disclosure is a PEG- didecyloxypropyl (CIO) conjugate, a PEG-dilauryloxypropyl (C12) conjugate, a PEG- dimyristyloxypropyl (C14) conjugate, a PEG-dipalmityloxypropyl (C16) conjugate, or a PEG- distearyloxypropyl (C18) conjugate. In certain of such embodiments, the PEG moiety has an average molecular weight of about 750 or about 2,000 daltons. In certain of such embodiments, the terminal hydroxyl group of the PEG moiety is substituted with a methyl group.

[1099] In addition to the foregoing, other hydrophilic polymers can be used in place of PEG. Examples of suitable polymers that can be used in place of PEG include, but are not limited to, polyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline, polyhydroxy propyl. methacrylamide, polymethacrylamide, and poly dimethylacrylamide, polylactic acid, polyglycolic acid, and derivatized celluloses such as hydroxymethylcellulose or hydroxyethylcellulose.

[1100] The lipids described herein can be combined in any number of molar ratios to produce an LNP. In certain embodiments, the LNP comprises a PEG-lipid where the PEG-lipid comprises at least 0.1 mol% of the total lipid. For example, but not by way of limitation, the PEG-lipid component can comprise from about 0. 1 mol% to 5 mol% of the total lipid. In certain embodiments, the LNP comprises a cationic lipid where the cationic lipid component comprises at least 10 mol% of the total lipid. For example, but not by way of limitation, the cationic lipid component can comprise from about 10 mol% to 70 mol% of the total lipid. In certain embodiments, the cationic lipid component can comprise from about 10 mol% to 60 mol% of the total lipid. In certain embodiments, the cationic lipid component can comprise from about 10 mol% to 50 mol% of the total lipid. In certain embodiments, the cationic lipid component can comprise from about 10 mol% to 40 mol% of the total lipid. In certain embodiments, the cationic lipid component can comprise from about 10 mol% to 30 mol% of the total lipid, the cationic lipid component can compnse from about 10 mol% to 20 mol% of the total lipid. In certain embodiments, the LNP comprises cholesterol where cholesterol comprises at least 10 mol% of the total lipid. For example, but not by way of limitation, cholesterol can comprise from about 10 mol% to 70 mol% of the total lipid. In certain embodiments, the cholesterol can comprise from about 10 mol% to 60 mol% of the total lipid. In certain embodiments, the cholesterol can comprise from about 10 mol% to 50 mol% of the total lipid. In certain embodiments, the cholesterol can comprise from about 10 mol% to 40 mol% of the total lipid. In certain embodiments, the cholesterol can comprise from about 10 mol% to 30 mol% of the total lipid, the cholesterol can comprise from about 10 mol% to 20 mol% of the total lipid. In certain embodiments, the LNP comprises anon-cationic lipid where the non-cationic lipid comprises at least 10 mol% of the total lipid. For example, but not by way of limitation, the non-cationic lipid can comprise from about 10 mol% to 70 mol% of the total lipid. In certain embodiments, the non-cationic lipid component can comprise from about 10 mol% to 60 mol% of the total lipid. In certain embodiments, the non-cationic lipid component can comprise from about 10 mol% to 50 mol% of the total lipid. In certain embodiments, the non-cationic lipid component can comprise from about 10 mol% to 40 mol% of the total lipid. In certain embodiments, the non-cationic lipid component can comprise from about 10 mol% to 30 mol% of the total lipid, the non-cationic lipid component can comprise from about 10 mol% to 20 mol% of the total lipid. In some embodiments, non-viral vectors include targeting modifications to improve uptake and / or selectively target certain cell types. These targeting modifications can include e.g., cell specific antigens, monoclonal antibodies, single chain antibodies, aptamers, polymers, sugars (e.g., N-acetylgalactosamine (GalNAc)), and cell penetrating peptides. Such vectors also optionally use fusogenic and endosome-destabilizing peptides / polymers, undergo acid- triggered conformational changes (e.g., to accelerate endosomal escape of the cargo), and / or incorporate a stimuli-cleavable polymer, e.g.. for release in a cellular compartment. For example, disulfide-based cationic polymers that are cleaved in the reducing cellular environment can be used.

[1101] In certain embodiments, one or more nucleic acid molecules (e.g., DNA molecules) other than the components of a genome editing system, e.g. the RNA-guided nuclease component (or a polynucleotide encoding the RNA-guided nuclease component) and / or the gRNA component described herein, are delivered. In certain embodiments, the nucleic acid molecule is delivered at the same time as one or more of the components of the genome editing system. In certain embodiments, the nucleic acid molecule is delivered before or after (e.g. , less than about 30 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 9 hours. 12 hours, 1 day, 2 days, 3 days, 1 week, 2 weeks, or 4 weeks) one or more of the components of the genome editing system are delivered. In certain embodiments, the nucleic acid molecule is delivered by a different means than one or more of the components of the genome editing system, e.g., the RNA-guided nuclease component (or a polynucleotide encoding the RNA-guided nuclease component) and / or the gRNA component, are delivered. The nucleic acid molecule can be delivered by any of the delivery methods described herein. For example, the nucleic acid molecule can be delivered by a viral vector, e.g. , an AAV or an integration-deficient lentivirus, and the RNA-guided nuclease molecule component (or a polynucleotide encoding the RNA- guided nuclease component) and / or the gRNA component can be delivered by electroporation, e.g., such that the toxicity caused by nucleic acids (e g., DNAs) can be reduced. In certain embodiments, the nucleic acid molecule encodes a therapeutic protein, e.g. , a protein described herein. In certain embodiments, the nucleic acid molecule comprises an RNA molecule, e.g. , an RNA molecule described herein.

[1102] 7. 1.2 Naked Nucleic Acids

[1103] Nucleic acids encoding genome editing systems or components thereof can be delivered directly to cells as naked DNA or RNA, for instance by means of transfection or electroporation, or can be conjugated to molecules (e.g., N-acetylgalactosamine) promoting uptake by the target cells (e.g., liver cells). Nucleic acid vectors, such as the vectors summarized in Table 11, can also be used. In some embodiments the genome editing system of the disclosure is delivered into cells by electroporation.

[1104] 7.1.3 Shuttle vectors

[1105] One approach for cell therapy processes includes the direct delivery of active proteins into human cells. A protein delivery agent, the Feldan Shuttle, is a protein-based deli very agent, which is designed for cell therapy (Del'Guidice et al., PloSOne. 2018 Apr 4;13(4):e0195558; incorporated in its entirety herein by reference). In some embodiments the genome editing system of the disclosure are delivered into cells by the Feldan Shuttle.

[1106] 7.1.4 Nucleic acid vectors

[1107] Nucleic acid vectors can comprise one or more sequences encoding genome editing system components, such as an RNA-guided nuclease, a gRNA and / or a donor template. A vector can also comprise a sequence encoding a signal peptide (e.g, for nuclear localization, nucleolar localization, or mitochondrial localization), associated with (e.g., inserted into or fused to) a sequence coding for a protein. As one example, a nucleic acid vectors can include an RNA-guided nuclease (e.g., Cas9 or Cas12a) coding sequence that encodes one or more nuclear localization sequences (e.g. a nuclear localization sequence from SV40).

[1108] The nucleic acid vector can also include any suitable number of regulatory / control elements, e.g, promoters, enhancers, introns, polyadenylation signals, Kozak consensus sequences, or internal ribosome entry sites (IRES). These elements are well known in the art and are described in Cotta-Ramusino.

[1109] Nucleic acid vectors according to this disclosure include recombinant viral vectors. Exemplary viral vectors are set forth in Table 12, and additional suitable viral vectors and their use and production are described in Cotta-Ramusino. Other viral vectors known in the art can also be used. In addition, viral particles can be used to deliver genome editing system components in nucleic acid and / or peptide form. For example, “empty7’ viral particles can be assembled to contain any suitable cargo. Viral vectors and viral particles can also be engineered to incorporate targeting ligands to alter target tissue specificity.

[1110] 7.2 Delivery of RNPs and / or RNA encoding genome editing system components

[1111] RNPs (complexes of gRNAs and RNA-guided nucleases) and / or RNAs encoding RNA- guided nucleases and / or gRNAs, can be delivered into cells or administered to subjects by art- known methods, some of which are described in Cotta-Ramusino. In vitro, RNA-guided nuclease-encoding and / or gRNA-encoding RNA can be delivered, e.g., by microinjection, electroporation, transient cell compression or squeezing (see, e.g., Lee 2012). Lipid-mediated transfection, peptide-mediated delivery, GalNAc- or other conjugate-mediated delivery, and combinations thereof, can also be used for delivery in vitro and in vivo.

[1112] In vitro, delivery via electroporation comprises mixing the cells with the RNA encoding RNA-guided nucleases and / or gRNAs, with or without donor template nucleic acid molecules, in a cartridge, chamber or cuvette and applying one or more electrical impulses of defined duration and amplitude. Systems and protocols for electroporation are known in the art, and any suitable electroporation tool and / or protocol can be used in connection with the various embodiments of this disclosure.

[1113] In certain embodiments, a genome editing system or ribonucleoprotein (RNP) complex, may comprise guide RNAs, Casl2a proteins, including modified Casl2a proteins (e.g., AsCasl2a variants). Non-limiting examples of Casl2a proteins are set forth in SEQ ID Nos: 71-79. In certain embodiments, a genome editing system or RNP complex may include a guide RNA (gRNA) complexed to a Casl2a protein or a modified Casl2a protein. In certain embodiments a gRNA may comprise a sequence set forth in SEQ ID Nos: 39-53 or SEQ ID Nos: 54-68. In certain embodiments, a genome editing system or RNP complex may comprise an RNP complex set forth in Table 15. For example, a genome editing system or RNP complex may include a gRNA comprising a sequence set forth in SEQ ID Nos: 39, 54, or 70, a modified Casl2a protein set forth In SEQ ID NO: 75 and target an LPA gene at the sequence set forth in SEQ ID NO: 24. In another example, a genome editing system or RNP complex may include a gRNA comprising a sequence set forth in SEQ ID Nos: 41, 56, or 70, a modified Casl2a protein set forth in SEQ ID NO: 75, and target an LPA gene at the sequence set forth in SEQ ID NO: 26.

[1114] In certain embodiments, a genome editing system or ribonucleoprotein (RNP) complex, may comprise guide RNAs, Casl2a proteins, including modified Casl2a proteins (e.g., AsCasl2a variants). Non-limiting examples of Casl2a proteins are set forth in SEQ ID NOs: 71-79. In certain embodiments, a genome editing system or RNP complex may include a guide RNA (gRNA) complexed to a Casl2a protein or a modified Casl2a protein. In certain embodiments a gRNA may comprise a sequence set forth in SEQ ID NOs: 336-346 or SEQ ID NOs: 362-372. In certain embodiments, a genome editing system or RNP complex may comprise an RNP complex set forth in Table 15. For example, a genome editing system or RNP complex may include a gRNA comprising a sequence set forth in SEQ ID NOs: 338, 364, or 394, a modified Casl2a protein set forth In SEQ ID NO: 75 and target w ANGPTL3 gene at the sequence set forth in SEQ ID NO: 312. In another example, a genome editing system or RNP complex may include a gRNA comprising a sequence set forth in SEQ ID NOs: 343, 369, or 394, a modified Casl2a protein set forth In SEQ ID NO: 75 and target m ANGPTI.3 gene at the sequence set forth in SEQ ID NO: 317.

[1115] In certain embodiments, a genome editing system or ribonucleoprotein (RNP) complex, may comprise guide RNAs, Casl2a proteins, including modified Casl2a proteins (e.g., AsCasl2a variants). Non-limiting examples of Casl2a proteins are set forth in SEQ ID NOs: 71-79. In certain embodiments, a genome editing system or RNP complex may include a guide RNA (gRNA) complexed to a Cas12a protein or a modified Cas12a protein. In certain embodiments a gRNA may comprise a sequence set forth in SEQ ID NOs: 347-361 or SEQ ID NOs: 373-387. In certain embodiments, a genome editing system or RNP complex may comprise an RNP complex set forth in Table 15. For example, a genome editing system or RNP complex may include a gRNA comprising a sequence set forth in SEQ ID NOs: 360, 386, or 395, a modified Casl2a protein set forth In SEQ ID NO: 75 and target an PCSK9 gene at the sequence set forth in SEQ ID NO: 334. In another example, a genome editing system or RNP complex may include a gRNA comprising a sequence set forth in SEQ ID NOs: 361, 387, or 395, a modified Casl2a protein set forth In SEQ ID NO: 75 and target an ANGPTL3 gene at the sequence set forth in SEQ ID NO: 335.

[1116] In certain embodiments, a genome editing system comprises guide RNA and mRNA encoding the RNA-guided nuclease (Casl2a proteins, including modified Casl2a proteins (e.g., AsCasl2a variants)). Non-limiting examples of mRNAs encoding the RNA-guided nuclease are set forth in SEQ ID NOs: 93-97. Following delivery’ of the guide RNA and mRNA to the cell, the mRNA drives translation of the RNA-guided nucleases, leading to formation of the RNP complexes comprising a guide RNA (gRNA) and a Casl2a protein or a modified Cas12a protein. In certain embodiments, a gRNA can comprise a sequence set forth in SEQ ID NOs: 39-53 or SEQ ID NOs: 54-68. In certain embodiments, a genome editing system or RNP complex can comprise an RNP complex set forth in Table 15. For example, but not by way of limitation, a genome editing system or RNP complex can comprise a gRNA comprising a sequence set forth in SEQ ID NOs: 39, 54, or 70, a modified Casl2a protein set forth in SEQ ID NO: 75 and target an LPA gene at the sequence set forth in SEQ ID NO: 24. Additionally or alternatively, a genome editing system or RNP complex can comprise a gRNA comprising a sequence set forth in SEQ ID NOs: 41, 56, or 70, a modified Casl2a protein set forth in SEQ ID NO: 75, and target an LPA gene at the sequence set forth in SEQ ID NO: 26.

[1117] In certain embodiments, a genome editing system comprises guide RNA and mRNA encoding the RNA-guided nuclease (Casl2a proteins, including modified Casl2a proteins (e.g., AsCasl2a variants)). Non-limiting examples of mRNAs encoding the RNA-guided nuclease are set forth in SEQ ID NOs: 93-97. Following delivery' of the guide RNA and mRNA to the cell, the mRNA drives translation of the RNA-guided nucleases, leading to formation of the RNP complexes comprising a guide RNA (gRNA) and a Casl2a protein or a modified Cas12a protein. In certain embodiments a gRNA may comprise a sequence set forth in SEQ ID NOs: 336-346 or SEQ ID NOs: 362-372. In certain embodiments, a genome editing system or RNP complex may comprise an RNP complex set forth in Table 15. For example, a genome editing system or RNP complex may include a gRNA comprising a sequence set forth in SEQ ID NOs: 338, 364, or 394, a modified Casl2a protein set forth In SEQ ID NO: 75 and target an ANGPT 3 gene at the sequence set forth in SEQ ID NO: 312. In another example, a genome editing system or RNP complex may include a gRNA comprising a sequence set forth in SEQ ID NOs: 343, 369. or 394. a modified Casl2a protein set forth In SEQ ID NO: 75 and target an ANGPTL3 gene at the sequence set forth in SEQ ID NO: 317.

[1118] In certain embodiments, a genome editing system comprises guide RNA and mRNA encoding the RNA-guided nuclease (Casl2a proteins, including modified Casl2a proteins (e.g., AsCasl2a variants)). Non-limiting examples of mRNAs encoding the RNA-guided nuclease are set forth in SEQ ID NOs: 93-97. Following delivery’ of the guide RNA and mRNA to the cell, the mRNA drives translation of the RNA-guided nucleases, leading to formation of the RNP complexes comprising a guide RNA (gRNA) and a Casl2a protein or a modified Cas12a protein. In certain embodiments a gRNA may comprise a sequence set forth in SEQ ID NOs: 347-361 or SEQ ID NOs: 373-387. In certain embodiments, a genome editing system or RNP complex may comprise an RNP complex set forth in Table 15. For example, a genome editing system or RNP complex may include a gRNA comprising a sequence set forth in SEQ ID NOs: 360, 386, or 395, a modified Casl2a protein set forth In SEQ ID NO: 75 and target an PCSK9 gene at the sequence set forth in SEQ ID NO: 334. In another example, a genome editing system or RNP complex may include a gRNA comprising a sequence set forth in SEQ ID NOs: 361, 387, or 395, a modified Casl2a protein set forth In SEQ ID NO: 75 and target an ANGPTL3 gene at the sequence set forth in SEQ ID NO: 335.

[1119] Table 15; Exemplary ribonucleoprotein (RNP) Configuration

[1120] 7.3 Editing, efficiency

[1121] In certain embodiments, editing the gene of interest using the genome editing systems, the RNP complexes, or the LNP delivery systems described in this disclosure reduces expression of the gene product of the gene of interest by at least about 20% to about 100% when compared to control non-edited cells. In some embodiments, the gene product level is reduced by about at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% compared to control cells. In some embodiments, the gene product levels are reduced by at least about 50% to at least about 95% compared to control cells. In some embodiments, the gene product levels are reduced by at least about 90% compared to control cells. In some embodiments, the gene product levels are reduced by at least about 95% compared to control cells.

[1122] In certain embodiments, the genome editing systems, the RNP complexes, or the LNP deliver^' systems described in this disclosure result in editing of the gene of interest with an efficiency of at least 20%, 25%, 30%, 35%, 40%, 45%, 50%. 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%. For example, the genome editing systems, the RNP complexes, or the LNP delivery systems, when contacting a population of cells (e.g. hepatocytes), a tissue (e.g., the liver), or a subject, may edit at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%. 80%. 85%. 90%, 95% or 100% of the copies of the gene of interest in the cell, tissue, or subject.

[1123] In certain embodiments, editing the LPA gene using the genome editing systems, the RNP complexes, or the LNP delivery systems described in this disclosure reduces Apo(a) expression by at least about 20% to about 100% when compared to control non-edited cells. In some embodiments, the Apo(a) level is reduced by about at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% compared to control cells. In some embodiments, the Apo(a) levels are reduced by at least about 50% to at least about 95% compared to control cells. In some embodiments, the Apo(a) levels are reduced by at least about 90% compared to control cells. In some embodiments, the Apo(a) levels are reduced by at least about 95% compared to control cells.

[1124] In certain embodiments, the genome editing systems, the RNP complexes, or the LNP delivery systems described in this disclosure result in editing of the L A gene with an efficiency of at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%. 95% or 100%. For example, the genome editing systems, the RNP complexes, or the LNP deliver^' systems, when contacting a population of cells (e.g. hepatocytes), a tissue (e.g., the liver), or a subject, may edit at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the copies of the LPA gene in the cell, tissue, or subj ect.

[1125] In certain embodiments, editing the ANGPT1.3 gene using the genome editing systems, the RNP complexes, or the LNP delivery systems described in this disclosure reduces ANGPTL3 expression by at least about 20% to about 100% when compared to control nonedited cells. In some embodiments, the ANGPTL3 level is reduced by about at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% compared to control cells. In some embodiments, the ANGPTL3 levels are reduced by at least about 50% to at least about 95% compared to control cells. In some embodiments, the ANGPTL3 levels are reduced by at least about 90% compared to control cells. In some embodiments, the ANGPTL3 levels are reduced by at least about 95% compared to control cells.

[1126] In certain embodiments, the genome editing systems, the RNP complexes, or the LNP delivery systems described in this disclosure result in editing of the ANGPTL3 gene with an efficiency of at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%. For example, the genome editing systems, the RNP complexes, or the LNP delivery' systems, when contacting a population of cells (e.g. hepatocytes), a tissue (e.g., the liver), or a subject, may edit at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the copies of the ANGPTL3 gene in the cell, tissue, or subject.

[1127] In certain embodiments, editing the PCSK9 gene using the genome editing systems, the RNP complexes, or the LNP delivery’ systems described in this disclosure reduces PCSK9 expression by at least about 20% to about 100% when compared to control non-edited cells. In some embodiments, the PCSK9 level is reduced by about at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% compared to control cells. In some embodiments, the PCSK.9 levels are reduced by at least about 50% to at least about 95% compared to control cells. In some embodiments, the PCSK.9 levels are reduced by at least about 90% compared to control cells. In some embodiments, the PCSK9 levels are reduced by at least about 95% compared to control cells.

[1128] In certain embodiments, the genome editing systems, the RNP complexes, or the LNP delivery systems described in this disclosure result in editing of the PCSK9 gene with an efficiency of at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%. For example, the genome editing systems, the RNP complexes, or the LNP delivery systems, when contacting a population of cells (e.g. hepatocytes), a tissue (e.g., the liver), or a subject, may edit at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the copies of the PCSK9 gene in the cell, tissue, or subject.

[1129] In certain embodiments, editing a liver-expressed gene using the genome editing systems, the RNP complexes, or the LNP delivery systems described in this disclosure reduces gene expression by at least about 20% to about 100% when compared to control non-edited cells. In some embodiments, the liver-expressed-gene-product level is reduced by about at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% compared to control cells. In some embodiments, the liver-expressed-gene-product levels are reduced by at least about 50% to at least about 95% compared to control cells. In some embodiments, the liver-expressed-gene-product levels are reduced by at least about 90% compared to control cells. In some embodiments, the liver-expressed-gene-product levels are reduced by at least about 95% compared to control cells.

[1130] In certain embodiments, the genome editing systems, the RNP complexes, or the LNP delivery systems described in this disclosure result in editing of the target gene with an efficiency of at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%. For example, the genome editing systems, the RNP complexes, or the LNP delivery systems, when contacting a population of cells (e.g. hepatocytes), a tissue (e.g., the liver), or a subject, may edit at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the copies of the target gene in the cell, tissue, or subject.

[1131] Target cells

[1132] The genome editing systems, the RNP complexes, or the delivery systems described in this disclosure can be used to manipulate or alter a target cell, e.g., to edit or alter a target nucleic acid. In some embodiments, the genome editing system, the RNP complex, or the delivery system described in this disclosure, are used to edit target cells in a tissue, e g., edit or alter a target nucleic acid in cells that make up the tissue. The manipulating can occur, in various embodiments, in vivo or ex vivo. In certain embodiments, the target cells are cells involved in metabolism (e.g, lipid metabolism). For example, in one embodiment, the target cells are liver cells (e.g., hepatocytes, hepatic stellate cells, a Kupffer cells, or liver stem cells). In one embodiment the target cell is a hepatocyte. In certain embodiments, the target cell comprises a genomic edit that results in loss of function of a liver-expressed gene. In certain embodiments, the liver-expressed gene is involved in regulating lipid and / or cholesterol metabolism. In certain embodiments, decreasing the expression of the liver-expressed gene decreases the levels of circulating LDL. In certain embodiments, decreasing the expression of the liver-expressed gene decreases the levels of circulating Lp(a). In certain embodiments, the liver-expressed gene is LPA, ANGPTL3, PCSK9. LDLR. APOC2. APOC3. APOB. MTP, ANGPTL4, ANGPTL8, APOA5, ApoB. APOE. IDOL, NPC1L1, ASGRI, TM6SF2. GALNT2, LPL, MLXIPL, SORT1. TRIB1, MARC1, ABCG5, ABCG8, PNPLA3, TM6SF2, HFE, HMOX- 1, UGT1A1, STAP1, LDLRAP1, LMF-1, GP1HBP1, CYP27A1, LIPA or ATP7B. In certain embodiments, the target cell comprises a genomic edit that results in loss of function of LPA, ANGPTL3, and / or PCSK9. In certain embodiments, the target cell comprises a genomic edit that results in loss of function of LPA, ANGPTL3, or PCSK9. While not wishing to be bound by any particular theory, it is contemplated that reducing or disabling LPA, ANGPTL3, or PCSK9 reduces the risk of major adverse cardiovascular events (MACE) including, heart attack, stroke, aortic stenosis, peripheral vascular disease, and renal dysfunction. In some embodiments, reducing or disabling expression of the liver-expressed gene reduces hyperlipidemia and / or hypercholesterolemia. In some embodiments, reducing or disabling LPA, ANGPTL3, or PCSK9 expression reduces hyperlipidemia and / or hypercholesterolemia.

[1133] In some embodiments, editing the LPA gene in the liver cells using the genome editing system, the RNP complex, or the delivery system described in this disclosure reduces Apo(a) levels by at least about 20% to about 100% when compared to a control, non-edited liver cells. In some embodiments, the Apo(a) level is reduced by about at least 20%, 25%, 30%, 35%, 40%, 45%. 50%. 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%. 95% or 100% compared to control cells. In some embodiments, the Apo(a) levels are reduced by at least 50% compared to control cells. In some embodiments, the Apo(a) levels are reduced by at least 80% compared to control cells. In some embodiments, the Apo(a) levels are reduced by at least 85% compared to control cells. In some embodiments, the Apo(a) levels are reduced by at least 90% compared to control cells. In some embodiments, the Apo(a) levels are reduced by at least about 95% compared to control cells.

[1134] In some embodiments, editing the ANGPTL3 gene in the liver cells using the genome editing system, the RNP complex, or the delivery system described in this disclosure reduces ANGPTL3 levels by at least about 20% to about 100% when compared to a control, non-edited liver cells. In some embodiments, the ANGPTL3 level is reduced by about at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% compared to control cells. In some embodiments, the ANGPTL3 levels are reduced by at least 50% compared to control cells. In some embodiments, the ANGPTL3 levels are reduced by at least 80% compared to control cells. In some embodiments, the ANGPTL3 levels are reduced by at least 85% compared to control cells. In some embodiments, the ANGPTL3 levels are reduced by at least 90% compared to control cells. In some embodiments, the ANGPTL3 levels are reduced by at least about 95% compared to control cells.

[1135] In some embodiments, editing the PCSK9 gene in the liver cells using the genome editing system, the RNP complex, or the delivery system described in this disclosure reduces PCSK9 levels by at least about 20% to about 100% when compared to a control, non-edited liver cells. In some embodiments, the PCSK9 level is reduced by about at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% compared to control cells. In some embodiments, the PCSK9 levels are reduced by at least 50% compared to control cells. In some embodiments, the PCSK9 levels are reduced by at least 80% compared to control cells. In some embodiments, the PCSK9 levels are reduced by at least 85% compared to control cells. In some embodiments, the PCSK9 levels are reduced by at least 90% compared to control cells. In some embodiments, the PCSK9 levels are reduced by at least about 95% compared to control cells.

[1136] In some embodiments, editing the liver-expressed gene in the liver cells using the genome editing system, the RNP complex, or the delivery system described in this disclosure reduces the liver-expressed-gene-product levels by at least about 20% to about 100% when compared to a control, non-edited liver cells. In some embodiments, the liver-expressed-gene- product level is reduced by about at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%, 90%, 95% or 100% compared to control cells. In some embodiments, the liver-expressed-gene-product levels are reduced by at least 50% compared to control cells. In some embodiments, the liver-expressed-gene-product levels are reduced by at least 80% compared to control cells. In some embodiments, the liver-expressed-gene-product levels are reduced by at least 85% compared to control cells. In some embodiments, the liver- expressed-gene-product levels are reduced by at least 90% compared to control cells. In some embodiments, the liver-expressed-gene-product levels are reduced by at least about 95% compared to control cells.

[1137] Provided herein are methods of administering the genome editing system, the RNP complex, or the delivery sy stem described in this disclosure, to treat, prevent or reduce the risk of diseases, conditions, and disorders, including metabolic disorders. Non-limiting examples of such disease include, hyperlipidemia, hypercholesterolemia, atherosclerotic cardiovascular disease (ASCVD), heart attack, stroke, aortic stenosis, high blood pressure, peripheral vascular disease, diabetes, renal dysfunction, or a combination of these. In some embodiments, the genome editing system, the RNP complex, or the delivery system described in this disclosure is administered to a subject or patient having the particular disease or condition to be treated. In some aspects, the methods thereby treat, e.g., ameliorate one or more symptom of, the disease or condition.

[1138] In some embodiments, administering the genome editing system, the RNP complex, or the delivery system described in this disclosure reduces Apo(a) levels (e.g., an Apo(A) expression level in a cell, or a serum or plasma Apo(a) levels) in the subject or patient to <150 mg / dL. In some embodiments, administering the genome editing system, the RNP complex, or the delivery system described in this disclosure reduces Apo(a) levels in the subject or patient to between about 5 mg / dL and about 140 mg / dL. In some embodiments, administering the genome editing system, the RNP complex, or the deliver)’ system described in this disclosure reduces Apo(a) levels in the subject or patient to between about 60 mg / dL and about 100 mg / dL, between about 70 mg / dL and about 100 mg / dL, between about 80 mg / dL and about 100 mg / dL, between about 90 mg / dL and about 100 mg / dL, between about 50 mg / dL and about 80 mg / dL, between about 30 mg / dL and about 50 mg / dL, between about 40 mg / dL and about 55 mg / dL, between about 40 mg / dL and about 60 mg / dL. between about 50 mg / dL and about 60 mg / dL, or between about 60 mg / dL and about 75 mg / dL. In some embodiments, administering the genome editing system, the RNP complex, or the delivery system described in this disclosure reduces Apo(a) levels in the subject or patient to between about 5 mg / dL and about 10 mg / dL, between about 7 mg / dL and about 15 mg / dL. between about 20 mg / dL and about 30 mg / dL, between about 25 mg / dL and about 40 mg / dL, or between about 30 mg / dL and about 45 mg / dL. In some embodiments, administering the genome editing system, the RNP complex, or the deliver)’ system described in this disclosure reduces Apo(a) levels in the subj ect or patient to less than 10 mg / dL. In some embodiments, administering the genome editing system, the RNP complex, or the delivery system described in this disclosure reduces Apo(a) levels in the subject or patient to between about 66 mg / dL and about 100 mg / dL. In some embodiments, administering the genome editing system, the RNP complex, or the delivery system described in this disclosure reduces Apo(a) levels in the subject or patient to between about 30 mg / dL and about 50 mg / dL. In some embodiments, administering the genome editing system, the RNP complex, or the delivery system described in this disclosure reduces an Apo(a) level (e.g., serum or plasma Apo(a) level) in the subject by about at least 20%, 25%, 30%, 35%, 40%, 45%. 50%. 55%. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% relative to the Apo(a) level in the subject prior to administration, (or relative to a control Apo(a) level). In some embodiments, administering the genome editing system, the RNP complex, or the delivery system described in this disclosure reduces an Apo(a) level (e.g., an Apo(A) expression level in a cell, or a serum or plasma Apo(a) level) in the subject by from about 20% to about 100%. from about 20% to about 90%. from about 20% to about 80%. from about 20% to about 70%, from about 20% to about 60%, from about 20% to about 40%, from about 40% to about 100%, from about 40% to about 90%, from about 40% to about 80%, from about 40% to about 70%, from about 40% to about 60%, from about 60% to about 100%, from about 60% to about 90%, from about 60% to about 80%, from about 60% to about 70%. from about 70% to about 100%, from about 70% to about 90%, from about 70% to about 80%, from about 80% to about 100%, from about 80% to about 90% or from about 90% to about 100% relative to the Apo(a) level in the subject prior to administration (or relative to a control Apo(a) level). In some embodiments, the Apo(a) level in the subject is measured at about 1, 2, 3, 4, 5, 6. 7, 8, 12. 16. or 20 weeks, about 1. 2, 3. 4, 5. or 6 months, or about 1, 2. 3, 4. or 5 years after administering the genome editing system, the RNP complex, or the delivery system described in this disclosure.

[1139] In some embodiments, administering the genome editing system, the RNP complex, or the deli...

Claims

WHAT IS CLAIMED IS:

1. A genome editing system comprising:(a) a gRNA molecule comprising a targeting domain that targets a target sequence of a gene of interest, wherein the gRNA molecule contains one or more modifications; and(b) an RNA-guided nuclease, or an RNA encoding the RNA-guided nuclease.

2. The genome editing system of claim 1, wherein the gRNA molecule further comprises a nucleotide extension, wherein the nucleotide extension is a 5’ extension, a 3’ extension, or any combination thereof.

3. The genome editing system of claim 1 or claim 2, wherein the one or more modifications is selected from the group consisting of a 5' inverted thymidine (idT) modification, a 3’ idT modification, a 2’ fluoro modification, a 2’ O-methyl modification, a phosphorothioate linkage, a 3’ pseudoknot, a locked nucleic acid (LN A), and any combination thereof.

4. The genome editing system of claim 3, wherein the one or more modifications comprises the 5’ inverted thymidine (idT) modification and the 3’ idT modification.

5. The genome editing system of claim 3 or claim 4, wherein the one or more modifications comprises the 2’ fluoro modification.

6. The genome editing system of any one of claims 3-5, wherein the one or more modifications comprises one or more 2’ fluoro modifications, and each of the 2‘ fluoro modifications modifies a nucleotide internal to the gRNA molecule.

7. The genome editing system of any one of claims 2-6, wherein the gRNA molecule further comprises a 5’ DNA extension.

8. The genome editing system of any one of claims 1 -7, wherein the RNA-guided nuclease comprises AsCasl2a.

9. The genome editing system of claim 8, wherein the gRNA molecule comprises a hairpin region capable of binding to the AsCasl2a.

10. The genome editing system of claim 9, wherein the one or more modifications of the gRNA are in the hairpin region, the targeting domain, or both.

11. The genome editing system of claim 10, wherein the hairpin region comprises SEQ ID NO: 252.

12. The genome editing system of claim 1 1, wherein the hairpin region comprises SEQ ID NO: 421.

13. The genome editing system of claim 11, wherein the hairpin region comprises SEQ ID NO: 427.

14. The genome editing system of any one of claims 9-13, wherein the gRNA molecule comprises a DNA extension 5’ to the hairpin region.

15. The genome editing system of any one of claim 9-14, wherein the one or more modifications comprises a 5’ inverted thymidine (idT) modification and / or a 3’ idT modification.

16. The genome editing system of any one of claims 1-15, wherein the one or more modifications of the gRNA enhance binding affinity of the gRNA molecule to the Cast 2a nuclease.17 The genome editing system of any one of claims 10-16, wherein the hairpin region is upstream of the targeting domain.

18. The genome editing system of any one of claims 10-17, wherein the hairpin region comprises the one or more modifications.

19. The genome editing system of any one of claims 1-18, wherein the one or more modifications include a 2’ fluoro modification.

20. The genome editing system of claim 19, w herein at least one of nucleotides 1, 5, 6, 7, 8, 9, 10, 12. 13, 14, 16, 17, 18 or 19 of the hairpin region is modified with a 2’ fluoro modification.

21. The genome editing system of claim 19, wherein each of nucleotides 1, 5, 6, 7, 8, 9, 10, 12, 13, 14, 16, 17, 18 and 19 of the hairpin region is modified with a 2' fluoro modification.

22. The genome editing system of claim 19, wherein a pattern of 2’ fluoro modifications on the hairpin region of the gRNA consists of 2’ fluoro modifications at nucleotides 1, 5, 6, 7,8, 9, 10, 12, 13, 14, 16, 17, 18 and 19 of the hairpin region.

23. The genome editing system of claim 19 or 20, wherein nucleotides 7 and 8 of the hairpin region are not modified with a 2’ fluoro modification.

24. The genome editing system of claim 19, 20, or 23, wherein each of nucleotides 1, 5, 6,9, 10, 12, 13, 14, 16, 17, 18 and 19 of the hairpin region is modified with a 2' fluoro modification.

25. The genome editing system of claim 19, 20, 23, or 24 wherein a pattern of 2’ fluoro modifications on the hairpin region of the gRNA consists of 2’ fluoro modifications at nucleotides 1, 5, 6. 9, 10, 12.

13.

14. 16, 17, 18 and 19 of the hairpin region.

26. The genome editing system of any one of claims 1-25, wherein the gene of interest is expressed in a liver cell of a subject.

27. A ribonucleoprotein (RNP) complex comprising the genome editing system of any one of claims 1-26.

28. A delivery system for delivering the genome editing system of any one of claims 1-26, wherein the delivery system comprises a DNA sequence encoding the gRNA molecule and / or RNA-guided nuclease, an RNA sequence encoding the gRNA molecule and / or RNA-guided nuclease, or any combination thereof.

29. The delivery system of claim 28, wherein the delivery system comprises a lipid nanoparticle (LNP) encapsulating the gRNA molecule and an RNA sequence encoding the RNA-guided nuclease.

30. The delivery system of claim 29, wherein the LNP comprises an ionizable lipid, a PEG lipid, a helper lipid, a sterol, or any combination thereof.31 . The delivery system of claim 30, wherein the ionizable lipid is selected from the group consisting of ((4-Hydroxybutyl)azanediyl)bis(hexane-6,l-diyl) bis(2 -hexyldecanoate) (ALC- 0315), 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl] amino] -octanoic acid, I -octylnonyl ester (SM-102), (2Z)-2-Nonen-l-yl 4-[[[[2-(dimethylamino)ethyl]thio]carbonyl][4-[(l- heptyloctyl)oxy]-4-oxobutyl] amino] butanoate (ATX-081), ATX-095 and ATX-0126.

32. The delivery system of claim 30 or claim 31, wherein the PEG lipid is selected from the group consisting of dimyristoyl-sn-glycero-3-methoxypolyethylene glycol (DMG-PEG); distearoyl-sn-glycerol-3-methoxypolyethylene glycol (DSG-PEG). and distearoyl-sn-glycero- 3-phosphoethanolamine-N-methoxypolyethylene glycol (DSPE-PEG).

33. The delivery system of any one of claims 30-32, wherein the helper lipid is selected from the group consisting of distearoylphosphatidylcholine (DSPC) and 1,2-dioleoyl-sn- glycero-3-phosphoethanolamine (DOPE).

34. The delivery' system of any one of claims 30-33, wherein the sterol is selected from the group consisting of cholesterol and sitosterol.35 The delivery^ system of claim 29, wherein the LNP comprises, ((4- Hydroxybutyl)azanediyl)bis(hexane-6,l-diyl) bis(2-hexyldecanoate) (ALC-0315), DMG- PEG, DSPC, and cholesterol.

36. A method of editing a gene of interest in a target cell comprising contacting the target cell with the genome editing system of any one of claims 1 -26, the RNP complex of claim 27, or the delivery system of any one of claims 28-35.

37. The method of claim 36, wherein the target cell is in vivo.

38. The method of claim 36 or claim 37, wherein the target cell is a cell involved in metabolism.

39. The method of any one of claims 36-38, wherein the target cell is a hepatocyte.

40. The method of any one of claims 36-39. wherein the gene of interest is edited with an editing efficiency of at least about 50%, 60%, 70%, 80%, 90% or about 100%.

41. A method of editing a gene of interest in a population of cells comprising contacting the population of cells with the genome editing system of any one of claims 1-26, the RNP complex of claim 27, or the delivery system of any one of claims 28-35.

42. The method of claim 41, wherein the population of cells is in vivo.

43. The method of claim 41 or 42, wherein at least about 50%, 60%, 70%, 80%, 90% or about 100% of the copies of the gene of interest in the population of cells are edited.

44. A method of editing a gene of interest in a subject comprising contacting the subject (or a cell or tissue from the subject) with the genome editing system of any one of claims 1-26, the RNP complex of claim 27, or the delivery system of any one of claims 28-35.

45. The method of claim 44, wherein the contacting occurs in vivo.

46. The method of claim 44 or 45, wherein at least about 50%, 60%, 70%, 80%, 90% or about 100% of the copies of the gene of interest in the subject, or in a target tissue or tissues in the subject, are edited.47 A method of treating a disease or disorder comprising administering to a subj ect in need thereof the genome editing system of any one of claims 1-26, the RNP complex of claim 27, or the delivery system of any one of claims 28-35.

48. The method of claim 47. wherein the disease or disorder is a hyperlipidemia or hypercholesterolemia.

49. The method of claim 47 or 48, wherein the disease or disorder is homozygous familial hypercholesterolemia (HoFH) or heterozygous familial hypercholesterolemia (HeFH).

50. The method of any one of claims 47-49, wherein the subject is suffering from an atherosclerotic cardiovascular disease (ASCVD).51 . The method of any one of claims 47-50, wherein the subject is identified to be at a high risk for a major adverse cardiovascular event (MACE) or has suffered from a MACE.

52. The method of any one of claims 47-51, wherein administering the genome editing system, the RNP complex, or the delivery system reduces a level of the gene of interest (or a gene product of the gene of interest) in the subject, or in a cell, tissue, or fluid from the subject, by at least about 50% to at least about 95% relative to the level prior to administration.

53. The method of any one of claims 47-52. wherein administering the genome editing system, the RNP complex, or the delivery system reduces an expression level of the gene of interest in a cell from the subject by at least about 50% to at least about 95% relative to the expression level prior to administration.

54. The method of any one of claims 47-53, wherein administering the genome editing system, the RNP complex, or the delivery system reduces an expression level of the gene of interest in a cell from the subject by at least 90% relative to the expression level prior to administration.

55. The method of any one of claims 47-54, wherein administering the genome editing system, the RNP complex, or the delivery system reduces a serum or plasma level of a gene product of the gene of interest in the subject by at least about 50% to at least about 95% relative to the serum or plasma level prior to administration.

56. The method of any one of claims 47-55, wherein administering the genome editing system, the RNP complex, or the delivery system reduces a serum or plasma level of a gene product of the gene of interest in the subject by at least about 50% to at least about 95% relative to the serum or plasma level prior to administration.

57. A method of treating a disease in a subject in need thereof, the method comprising administering to the subject a formulation comprising:(i) a lipid nanoparticle (LNP);(ii) an mRNA encoding a Casl 2a nuclease; and(iii) a gRNA molecule comprising a targeting domain that targets a sequence of a gene of interest of the subject, wherein the gRNA molecule contains one or more modifications; and wherein the mRNA and the gRNA are encapsulated within the LNP.58 The method of claim 57, wherein the gRNA molecule further comprises a nucleotide extension, wherein the nucleotide extension is a 5' extension, a 3’ extension, or any combination thereof.

59. The method of claim 57 or claim 58, wherein the one or more modifications is selected from the group consisting of a 5‘ inverted thymidine (idT) modification, a 3’ idT modification, a 2' fluoro modification, a 2’ O-methyl modification, a phosphorothioate linkage, a 3’ pseudoknot, a locked nucleic acid (LNA), and any combination thereof.

60. The method of claim 59, wherein the one or more modifications comprises the 5’ inverted thymidine (idT) modification and the 3’ idT modification.

61. The method of claim 59 or claim 60, wherein the one or more modifications comprises the 2’ fluoro modification.

62. The method of any one of claims 57-61, wherein the one or more modifications comprises one or more 2’ fluoro modifications, and each of the 2’ fluoro modifications modifies a nucleotide internal to the gRNA molecule.

63. The method of any one of claims 57-62, wherein the gRNA molecule further comprises a 5 ’ DNA extension.

64. The method of any one of claims 57-63, wherein the gRNA molecule comprises a hairpin region capable of binding to the Cas12a.

65. The method of any one of claims 57-64, wherein the Casl2a is AsCasl2a.

66. The method of claim 64 or 65, wherein the one or more modifications of the gRNA are in the hairpin region, the targeting domain, or both.

67. The method of claim 66, wherein the hairpin region comprises SEQ ID NO: 252.

68. The method of claim 66, wherein the hairpin region comprises SEQ ID NO: 421.

69. The method of claim 66, wherein the hairpin region comprises SEQ ID NO: 427.

70. The method of any one of claims 57-69, wherein the gRNA molecule comprises a DNA extension 5 ’ to the hairpin region.

71. The method of any one of claim 57-70, wherein the one or more modifications comprises a 5’ inverted thymidine (idT) modification and / or a 3’ idT modification.

72. The method of any one of claims 57-71, wherein the one or more modifications of the gRNA enhance binding affinity of the gRNA molecule to the Cas12a nuclease.

73. The method of any one of claims 64-72, wherein the hairpin region is upstream of the targeting domain.

74. The method of claim 73, wherein the hairpin region comprises one or more modifications.

75. The method of claim 74, wherein the one or more modifications include a 2’ fluoro modification.

76. The method of claim 75, wherein at least one of nucleotides 1, 5, 6, 7, 8, 9, 10, 12, 13, 14, 16, 17, 18 or 19 of the hairpin region is modified with a 2’ fluoro modification.

77. The method of claim 75, wherein each of nucleotides 1, 5, 6, 7, 8, 9, 10, 12, 13, 14, 16, 17, 18 and 19 of the hairpin region is modified w ith a 2' fluoro modification.

78. The method of claim 75, wherein a pattern of 2’ fluoro modifications on the hairpin region of the gRNA consists of 2’ fluoro modifications at nucleotides 1 , 5, 6, 7, 8, 9, 10, 12,13, 14, 16, 17, 18 and 19 of the hairpin region.

79. The method of claim 75 or 76, wherein nucleotides 7 and 8 of the hairpin region are not modified with a 2’ fluoro modification.

80. The method of claim 75, 76, or 79, wherein each of nucleotides 1, 5, 6, 9, 10, 12, 13,14, 16, 17, 18 and 19 of the hairpin region is modified with a 2’ fluoro modification.

81. The method of claim 75.

76.

79. or 80 wherein a pattern of 2’ fluoro modifications on the hairpin region of the gRNA consists of 2’ fluoro modifications at nucleotides 1 , 5, 6, 9, 10, 12, 13, 14, 16, 17, 18 and 19 of the hairpin region.

82. The method of any one of claims 57-81, wherein the gene of interest is expressed in a liver cell of a subject.

83. The method of claim 57, wherein the disease is a hyperlipidemia or hypercholesterolemia.

84. The method of claim 83, wherein the disease is homozygous familial hypercholesterolemia (HoFH) or heterozy gous familial hypercholesterolemia (HeFH).

85. A gRNA molecule comprising a targeting domain that targets a target sequence of a gene of interest, wherein the gRNA molecule comprises one or more modifications selected from the group consisting of a 5’ inverted thymidine (idT) modification, a 3’ idT modification, a 2’ fluoro modification, a 2’ O-methyl modification, a phosphorothioate linkage, a 3’ pseudoknot, a locked nucleic acid (LNA), and any combination thereof.

86. The gRNA molecule of claim 85, wherein the one or more modifications are on nucleotides positioned outside of the targeting domain.

87. The gRNA molecule of claim 85 or claim 86, wherein the gRNA comprises a 5’ DNA extension.

88. The gRNA molecule of any one of claims 85-87, wherein the 5’ DNA extension comprises the sequence set forth in SEQ ID NO: 7.

90. The gRNA molecule of claim 85, wherein the gRNA molecule comprises the following sequence: a. / 5InvdT / ATGTGTTTTTGTCAAAAGACCTTTT / i2FU / rArArU / i2FU / / i2FU / / i2FC / / i 2FU / / i2FA / / i2FC / rU / i2FC / / i2FU / / i2FU / rG / i2FU / / i2FA / / i2FG / / i2FA / rUx / 3InvdT / (SEQ ID NO: 113); b. / 5InvdT / ATGTGTTTTTGTCAAAAGACCTTTT / i2FU / rArArU / i2FU / / i2FU / / i2FC / / i 2FU / / i2FA / / i2FC / rU / i2FC / / i2FU / / i2FU / rG / i2FU / / i2FA / / i2FG / / i2FA / rUx (SEQ ID NO: 438); c. / i2FU / rArArU / i2FU / / i2FU / / i2FC / / i2FU / / i2FA / / i2FC / rU / i2FC / / i2FU / / i2FU / rG / i2FU / / i 2FA / / i2FG / / i2FA / rUx / 3InvdT / (SEQ ID NO: 439); or d. i2FU / rArArU / i2FU / / i2FU / / i2FC / / i2FU / / i2FA / / i2FC / rU / i2FC / / i2FU / / i2FU / rG / i2FU / / i 2FA / / i2FG / / i2FA / rUx (SEQ ID NO: 440); and wherein x consists of the targeting domain.

91. The gRNA molecule of claim 85, wherein the gRNA molecule comprises the following sequence: mA*TGTGTTTTTGTCAAAAGACCTTTT / i2FU / rArArU / i2FU / / i2FU / / i2FC / / i2FU / / i 2FA / / i2FC / rU / i2FC / / i2FU / / i2FU / rG / i2FU / / i2FA / / i2FG / / i2FA / rUx (SEQ ID NO: 114); wherein x consists of the targeting domain; and wherein the 3’ nucleotide of the targeting domain comprises a 2’ O-methyl modification and is linked to an adjacent nucleotide by a phosphorothioate linkage.

92. The gRNA molecule of claim 85, wherein the gRNA molecule comprises the following sequence: / 5InvdT / ATGTGTTTTTGTCAAAAGACCTTTTrUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUx / 3InvdT7 (SEQ ID NO: 115); wherein x consists of the targeting domain.

93. The gRNA molecule of claim 85, wherein the gRNA molecule comprises the following sequence: a. / 5InvdT / ATGTGTTTTTGTCAAAAGACCTTTT / i2FU / rArArU / i2FU / / i2FU / rCrU / i2 FA / / i2FC / rU / i2FC / / i2FU / / i2FU / rG / i2FU / / i2FA / / i2FG / / i2FA / rUx / 3InvdT / (SEQ ID NO: 116); b. / 5InvdT / ATGTGTTTTTGTCAAAAGACCTTTT / i2FU / rArArU / i2FU / / i2FU / rCrU / i2 FA / / i2FC / rU / i2FC / / i2FU / / i2FU / rG / i2FU / / i2FA / / i2FG / / i2FA / rUx (SEQ ID NO: 441); c. / i2FU / rArArU / i2FU / / i2FU / rCrU / i2FA / / i2FC / rU / i2FC / / i2FU / / i2FU / rG / i2FU / / i2FA / / i2 FG / / i2FA / rUx / 3InvdT / (SEQ ID NO: 442); or d. / i2FU / rArArU / i2FU / / i2FU / rCrU / i2FA / / i2FC / rU / i2FC / / i2FU / / i2FU / rG / i2FU / / i2FA / / i2 FG / / i2FA / rUx (SEQ ID NO: 443); and wherein x consists of the targeting domain.

94. The gRNA molecule of claim 85, wherein the gRNA molecule comprises the following sequence: a. mA*TGTGTTTTTGTCAAAAGACCTTTT / i2FU / rArArU / i2FU / / i2FU / rCrU / i2FA / / i2 FC / rU / i2FC / / i2FU / / i2FU / rG / i2FU / / i2FA / / i2FG / / i2FA / rUx (SEQ ID NO: 117); b. / 5InvdT / ATGTGTTTTTGTCAAAAGACCTTTTrUrArArU / i2FU / / i2FU / / i2FC / / i2FU / / i2FA / / i2FC / rU / i2FC / / i2FU / / i2FU / rG / i2FU / / i2FA / / i2FG / / i2FA / rUx / 3InvdT / (SEQ ID NO: 444) c. / 5InvdT / ATGTGTTTTTGTCAAAAGACCTTTTrUrArArU / i2FU / / i2FU / / i2FC / / i2FU / / i2FA / / i2FC / rU / i2FC / / i2FU / / i2FU / rG / i2FU / / i2FA / / i2FG / / i2FA / rUx (SEQ ID NO:445); d. / 5InvdT / ATGTGTTTTTGTCAAAAGACCTTTTrUrArArU / i2FU / / i2FU / rCrU / i2FA / / i2FC / rU / i2FC / / i2FU / / i2FU / rG / i2FU / / i2FA / / i2FG / / i2FA / rUx / 3InvdT / (SEQ ID NO:446); or e. 5InvdT / ATGTGTTTTTGTCAAAAGACCTTTTrUrArArU / i2FU / / i2FU / rCrU / i2FA / / i 2FC / rU / i2FC / / i2FU / / i2FU / rG / i2FU / / i2FA / / i2FG / / i2FA / rUx (SEQ ID NO: 447); and wherein x consists of the targeting domain; andwherein the 3’ nucleotide of the targeting domain comprises a 2’ O-methyl modification and is linked to an adjacent nucleotide by a phosphorothioate linkage.

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